Toxin peptide therapeutic agents

ABSTRACT

Disclosed is a composition of matter of the formula
 
(X 1 ) a —(F 1 ) d —(X 2 ) b —(F 2 ) e —(X 3 ) c   (I)
 
and multimers thereof, in which F 1  and F 2  are half-life extending moieties, and d and e are each independently 0 or 1, provided that at least one of d and e is 1; X 1 , X 2 , and X 3  are each independently -(L) f -P-(L) g -, and f and g are each independently 0 or 1; P is a toxin peptide of no more than about 80 amino acid residues in length, comprising at least two intrapeptide disulfide bonds; L is an optional linker; and a, b, and c are each independently 0 or 1, provided that at least one of a, b and c is 1. Linkage to the half-life extending moiety or moieties increases the in vivo half-life of the toxin peptide, which otherwise would be quickly degraded. A pharmaceutical composition comprises the composition and a pharmaceutically acceptable carrier. Also disclosed are a DNA encoding the inventive composition of matter, an expression vector comprising the DNA, and a host cell comprising the expression vector. Methods of treating an autoimmune disorder, such as, but not limited to, multiple sclerosis, type 1 diabetes, psoriasis, inflammatory bowel disease, contact-mediated dermatitis, rheumatoid arthritis, psoriatic arthritis, asthma, allergy, restinosis, systemic sclerosis, fibrosis, scleroderma, glomerulonephritis, Sjogren syndrome, inflammatory bone resorption, transplant rejection, graft-versus-host disease, and lupus and of preventing or mitigating a relapse of a symptom of multiple sclerosis are also disclosed.

This application claims the benefit of U.S. Provisional Application No.60/672,342, filed Apr. 22, 2005, which is hereby incorporated byreference.

This application incorporates by reference all subject matter containedon the compact disc, which is identified by the name of the file,A-1006.ST25.txt created on Apr. 17, 2006, the size of which file is 744KB.

Throughout this application various publications are referenced withinparentheses or brackets. The disclosures of these publications in theirentireties are hereby incorporated by reference in this application inorder to more fully describe the state of the art to which thisinvention pertains.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is related to the biochemical arts, in particularto therapeutic peptides and conjugates.

2. Discussion of the Related Art

Ion channels are a diverse group of molecules that permit the exchangeof small inorganic ions across membranes. All cells require ion channelsfor function, but this is especially so for excitable cells such asthose present in the nervous system and the heart. The electricalsignals orchestrated by ion channels control the thinking brain, thebeating heart and the contracting muscle. Ion channels play a role inregulating cell volume, and they control a wide variety of signalingprocesses.

The ion channel family includes Na⁺, K⁺, and Ca²⁺ cation and Cl⁻ anionchannels. Collectively, ion channels are distinguished as eitherligand-gated or voltage-gated. Ligand-gated channels include bothextracellular and intracellular ligand-gated channels. The extracellularligand-gated channels include the nicotinic acetylcholine receptor(nAChR), the serotonin (5-hdroxytryptamine, 5-HT) receptors, the glycineand γ-butyric acid receptors (GABA) and the glutamate-activated channelsincluding kanate, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid(AMPA) and N-methyl-D-aspartate receptors (NMDA) receptors. (Harte andOuzounis (2002), FEBS Lett. 514: 129-34). Intracellular ligand gatedchannels include those activated by cyclic nucleotides (e.g. cAMP,cGMP), Ca²⁺ and G-proteins. (Harte and Ouzounis (2002), FEBS Lett. 514:129-34). The voltage-gated ion channels are categorized by theirselectivity for inorganic ion species, including sodium, potassium,calcium and chloride ion channels. (Harte and Ouzounis (2002), FEBSLett. 514: 129-34).

A unified nomenclature for classification of voltage-gated channels wasrecently presented. (Catterall et al. (2000), Pharmacol. Rev. 55: 573-4;Gutman et al. (2000), Pharmacol. Rev. 55, 583-6; Catterall et al. (2000)Pharmacol. Rev. 55: 579-81; Catterall et al. (2000), Pharmacol. Rev. 55:575-8; Hofmann et al. (2000), Pharmacol. Rev. 55: 587-9; Clapham et al.(2000), Pharmacol Rev. 55: 591-6; Chandy (1991), Nature 352: 26; Goldinet al. (2000), Neuron 28: 365-8; Ertel et al. (2000), Neuron 25: 533-5).

The K⁺ channels constitute the largest and best characterized family ofion channels described to date. Potassium channels are subdivided intothree general groups: the 6 transmembrane (6TM) K⁺ channels, the2TM-2TM/leak K⁺ channels and the 2TM/Kir inward rectifying channels.(Tang et al. (2004), Ann. Rev. Physiol. 66, 131-159). These three groupsare further subdivided into families based on sequence similarity. Thevoltage-gated K⁺ channels, including (Kv1-6, Kv8-9), EAG, KQT, and Slo(BKCa), are family members of the 6TM group. The 2TM-2TM group comprisesTWIK, TREK, TASK, TRAAK, and THIK, whereas the 2TM/Kir group consists ofKir1-7. Two additional classes of ion channels include the inwardrectifier potassium (IRK) and ATP-gated purinergic (P2X) channels.(Harte and Ouzounis (2002), FEBS Lett. 514: 129-34).

Toxin peptides produced by a variety of organisms have evolved to targetion channels. Snakes, scorpions, spiders, bees, snails and sea anemoneare a few examples of organisms that produce venom that can serve as arich source of small bioactive toxin peptides or “toxins” that potentlyand selectively target ion channels and receptors. In most cases, thesetoxin peptides have evolved as potent antagonists or inhibitors of ionchannels, by binding to the channel pore and physically blocking the ionconduction pathway. In some other cases, as with some of the tarantulatoxin peptides, the peptide is found to antagonize channel function bybinding to a region outside the pore (e.g., the voltage sensor domain).

The toxin peptides are usually between about 20 and about 80 amino acidsin length, contain 2-5 disulfide linkages and form a very compactstructure (see, e.g., FIG. 10). Toxin peptides (e.g., from the venom ofscorpions, sea anemones and cone snails) have been isolated andcharacterized for their impact on ion channels. Such peptides appear tohave evolved from a relatively small number of structural frameworksthat are particularly well suited to addressing the critical issues ofpotency and stability. The majority of scorpion and Conus toxinpeptides, for example, contain 10-40 amino acids and up to fivedisulfide bonds, forming extremely compact and constrained structure(microproteins) often resistant to proteolysis. The conotoxin andscorpion toxin peptides can be divided into a number of superfamiliesbased on their disulfide connections and peptide folds. The solutionstructure of many of these has been determined by NMR spectroscopy,illustrating their compact structure and verifying conservation of theirfamily fold. (E.g., Tudor et al., lonisation behaviour and solutionproperties of the potassium-channel blocker ShK toxin, Eur. J. Biochem.251(1-2):133-41(1998); Pennington et al., Role of disulfide bonds in thestructure and potassium channel blocking activity of ShK toxin, Biochem.38(44): 14549-58 (1999); Jaravine et al., Three-dimensional structure oftoxin OSK1 from Orthochirus scrobiculosus scorpion venom, Biochem.36(6):1223-32 (1997); del Rio-Portillo et al.; NMR solution structure ofCn12, a novel peptide from the Mexican scorpion Centruroides noxius witha typical beta-toxin sequence but with alpha-like physiologicalactivity, Eur. J. Biochem. 271(12): 2504-16 (2004); Prochnicka-Chalufouret al., Solution structure of discrepin, a new K+-channel blockingpeptide from the alpha-KT×15 subfamily, Biochem. 45(6):1795-1804(2006)).

Conserved disulfide structures can also reflect the individualpharmacological activity of the toxin family. (Nicke et al. (2004), Eur.J. Biochem. 271: 2305-19, Table 1; Adams (1999), Drug Develop. Res. 46:219-34). For example, α-conotoxins have well-defined four cysteine/twodisulfide loop structures (Loughnan, 2004) and inhibit nicotinicacetylcholine receptors. In contrast, ω-conotoxins have sixcysteine/three disulfide loop consensus structures (Nielsen, 2000) andblock calcium channels. Structural subsets of toxins have evolved toinhibit either voltage-gated or calcium-activated potassium channels.FIG. 9 shows that a limited number of conserved disulfide architecturesshared by a variety of venomous animals from bee to snail and scorpionto snake target ion channels. FIG. 7 shows alignment of alpha-scorpiontoxin family and illustrates that a conserved structural framework isused to derive toxins targeting a vast array of potassium channels.

Due to their potent and selective blockade of specific ion channels,toxin peptides have been used for many years as tools to investigate thepharmacology of ion channels. Other than excitable cells and tissuessuch as those present in heart, muscle and brain, ion channels are alsoimportant to non-excitable cells such as immune cells. Accordingly, thepotential therapeutic utility of toxin peptides has been considered fortreating various immune disorders, in particular by inhibition ofpotassium channels such as Kv1.3 and IKCa1 since these channelsindirectly control calcium signaling pathway in lymphocytes. [e.g., Kemet al., ShK toxin compositions and methods of use, U.S. Pat. No.6,077,680; Lebrun et al., Neuropeptides originating in scorpion, U.S.Pat. No. 6,689,749; Beeton et al., Targeting effector memory T cellswith a selective peptide inhibitor of Kv1.3 channnels for therapy ofautoimmune diseases, Molec. Pharmacol. 67(4):1369-81 (2005); Mouhat etal., K⁺ channel types targeted by synthetic OSK1, a toxin fromOrthochirus scrobiculosus scorpion venom, Biochem. J. 385:95-104 (2005);Mouhat et al., Pharmacological profiling of Orthochirus scrobiculosustoxin 1 analogs with a trimmed N-terminal domain, Molec. Pharmacol.69:354-62 (2006); Mouhat et al., OsK1 derivatives, WO 2006/002850 A2; B.S. Jensen et al. The Ca²⁺-activated K+ Channel of IntermediateConductance: A Molecular Target for Novel Treatments?, Current DrugTargets 2:401-422 (2001); Rauer et al., Structure-guided Transformationof Charybdotoxin Yields an Analog That Selectively TargetsCa²⁺-activated over Voltage-gated K⁺ Channels, J. Biol. Chem. 275:1201-1208 (2000); Castle et al., Maurotoxin: A Potent Inhibitor ofIntermediate Conductance Ca²⁺-Activated Potassium Channels, MolecularPharmacol. 63: 409-418 (2003); Chandy et al., K⁺ channels as targets forspecific Immunomodulation, Trends in Pharmacol. Sciences 25: 280-289(2004); Lewis & Garcia, Therapeutic Potential of Venom Peptides, Nat.Rev. Drug Discov. 2: 790-802 (2003)].

Small molecules inhibitors of Kv1.3 and IKCa1 potassium channels and themajor calcium entry channel in T cells, CRAC, have also been developedto treat immune disorders [A. Schmitz et al. (2005) Molecul. Pharmacol.68, 1254; K. G. Chandy et al. (2004) TIPS 25, 280; H. Wulff et al.(2001) J. Biol. Chem. 276, 32040; C. Zitt et al. (2004) J. Biol. Chem.279, 12427], but obtaining small molecules with selectivity toward someof these targets has been difficult.

Calcium mobilization in lymphocytes is known to be a critical pathway inactivation of inflammatory responses [M. W. Winslow et al. (2003)Current Opinion Immunol. 15, 299]. Compared to other cells, T cells showa unique sensitivity to increased levels of intracellular calcium andion channels both directly and indirectly control this process. Inositoltriphosphate (IP3) is the natural second messenger which activates thecalcium signaling pathway. IP3 is produced following ligand-inducedactivation of the T cell receptor (TCR) and upon binding to itsintracellular receptor (a channel) causes unloading of intracellularcalcium stores. The endoplasmic reticulum provides one key calciumstore. Thapsigargin, an inhibitor of the sarcoplasmic-endoplasmicreticulum calcium ATPase (SERCA), also causes unloading of intracellularstores and activation of the calcium signaling pathway in lymphocytes.Therefore, thapsigargin can be used as a specific stimulus of thecalcium signaling pathway in T cells. The unloading of intracellularcalcium stores in T cells is known to cause activation of a calciumchannel on the cell surface which allows for influx of calcium fromoutside the cell. This store operated calcium channel (SOCC) on T cellsis referred to as “CRAC” (calcium release activated channel) andsustained influx of calcium through this channel is known to be criticalfor full T cell activation [S. Feske et al. (2005) J. Exp. Med. 202, 651and N. Venkatesh et al. (2004) PNAS 101, 8969]. For many years it hasbeen appreciated that in order to maintain continued calcium influx intoT cells, the cell membrane must remain in a hyperpolarized conditionthrough efflux of potassium ions. In T cells, potassium efflux isaccomplished by the voltage-gated potassium channel Kv1.3 and thecalcium-activated potassium channel IKCa1 [K. G. Chandy et al. (2004)TIPS 25, 280]. These potassium channels therefore indirectly control thecalcium signaling pathway, by allowing for the necessary potassiumefflux that allows for a sustained influx of calcium through CRAC.

Sustained increases in intracellular calcium activate a variety ofpathways in T cells, including those leading to activation of NFAT,NF-kB and AP-1 [Quintana-A (2005) Pflugers Arch. Eur. J. Physiol. 450,1]. These events lead to various T cell responses including alterationof cell size and membrane organization, activation of cell surfaceeffector molecules, cytokine production and proliferation. Severalcalcium sensing molecules transmit the calcium signal and orchestratethe cellular response. Calmodulin is one molecule that binds calcium,but many others have been identified (M. J. Berridge et al. (2003) Nat.Rev. Mol. Cell. Biol. 4,517). The calcium-calmodulin dependentphosphatase calcineurin is activated upon sustained increases inintracellular calcium and dephosphorylates cytosolic NFAT.Dephosphorylated NFAT quickly translocates to the nucleus and is widelyaccepted as a critical transcription factor for T cell activation (F.Macian (2005) Nat. Rev. Immunol. 5, 472 and N. Venkatesh et al. (2004)PNAS 101, 8969). Inhibitors of calcineurin, such as cyclosporin A(Neoral, SandImmune) and FK506 (Tacrolimus) are a main stay fortreatment of severe immune disorders such as those resulting inrejection following solid organ transplant (I. M. Gonzalez-Pinto et al.(2005) Transplant. Proc. 37, 1713 and D. R. J. Kuypers (2005) TransplantInternational 18, 140). Neoral has been approved for the treatment oftransplant rejection, severe rheumatoid arthritis (D. E. Yocum et al.(2000) Rheumatol. 39, 156) and severe psoriasis (J. Koo (1998) BritishJ. Dermatol. 139, 88). Preclinical and clinical data has also beenprovided suggesting calcineurin inhibitors may have utility in treatmentof inflammatory bowel disease (IBD; Baumgart D C (2006) Am. J.Gastroenterol. March 30; Epub ahead of print), multiple sclerosis (Ann.Neurol. (1990) 27, 591) and asthma (S. Rohatagi et al. (2000) J. Clin.Pharmacol. 40, 1211). Lupus represents another disorder that may benefitfrom agents blocking activation of helper T cells. Despite theimportance of calcineurin in regulating NFAT in T cells, calcineurin isalso expressed in other tissues (e.g. kidney) and cyclosporine A & FK506have a narrow safety margin due to mechanism based toxicity. Renaltoxicity and hypertension are common side effects that have limited thepromise of cyclosporine & FK506. Due to concerns regarding toxicity,calcineurin inhibitors are used mostly to treat only severe immunedisease (Bissonnette-R et al. (2006) J. Am. Acad. Dermatol. 54, 472).Kv1.3 inhibitors offer a safer alternative to calcineurin inhibitors forthe treatment of immune disorders. This is because Kv1.3 also operatesto control the calcium signaling pathway in T cells, but does so througha distinct mechanism to that of calcineurin inhibitors, and evidence onKv1.3 expression and function show that Kv1.3 has a more restricted rolein T cell biology relative to calcineurin, which functions also in avariety of non-lymphoid cells and tissues.

Calcium mobilization in immune cells also activates production of thecytokines interleukin 2 (IL-2) and interferon gamma (IFNg) which arecritical mediators of inflammation. IL-2 induces a variety of biologicalresponses ranging from expansion and differentiation of CD4⁺ and CD8⁺ Tcells, to enhancement of proliferation and antibody secretion by Bcells, to activation of NK cells [S. L. Gaffen & K. D. Liu (2004)Cytokine 28, 109]. Secretion of IL-2 occurs quickly following T cellactivation and T cells represent the predominant source of thiscytokine. Shortly following activation, the high affinity IL-2 receptor(IL2-R) is upregulated on T cells endowing them with an ability toproliferate in response to IL-2. T cells, NK cells, B cells andprofessional antigen presenting cells (APCs) can all secrete IFNg uponactivation. T cells represent the principle source of IFNg production inmediating adaptive immune responses, whereas natural killer (NK) cells &APCs are likely an important source during host defense againstinfection [K. Schroder et al. (2004) J. Leukoc. Biol. 75, 163]. IFNg,originally called macrophage-activating factor, upregulates antigenprocessing and presentation by monocytes, macrophages and dendriticcells. IFNg mediates a diverse array of biological activities in manycell types [U. Boehm et al. (1997) Annu. Rev. Immunol. 15, 749]including growth & differentiation, enhancement of NK cell activity andregulation of B cell immunoglobulin production and class switching.

CD40L is another cytokine expressed on activated T cells followingcalcium mobilization and upon binding to its receptor on B cellsprovides critical help allowing for B cell germinal center formation, Bcell differentiation and antibody isotype switching. CD40L-mediatedactivation of CD40 on B cells can induce profound differentiation andclonal expansion of immunoglobulin (Ig) producing B cells [S. Quezada etal. (2004) Annu. Rev. Immunol. 22, 307]. The CD40 receptor can also befound on dendritic cells and CD40L signaling can mediate dendritic cellactivation and differentiation as well. The antigen presenting capacityof B cells and dendritic cells is promoted by CD40L binding, furtherillustrating the broad role of this cytokine in adaptive immunity. Giventhe essential role of CD40 signaling to B cell biology, neutralizingantibodies to CD40L have been examined in preclinical and clinicalstudies for utility in treatment of systemic lupus erythematosis(SLE),—a disorder characterized by deposition of antibody complexes intissues, inflammation and organ damage [J. Yazdany and J Davis (2004)Lupus 13, 377].

Production of toxin peptides is a complex process in venomous organisms,and is an even more complex process synthetically. Due to theirconserved disulfide structures and need for efficient oxidativerefolding, toxin peptides present challenges to synthesis. Althoughtoxin peptides have been used for years as highly selectivepharmacological inhibitors of ion channels, the high cost of synthesisand refolding of the toxin peptides and their short half-life in vivohave impeded the pursuit of these peptides as a therapeutic modality.Far more effort has been expended to identify small molecule inhibitorsas therapeutic antagonists of ion channels, than has been given thetoxin peptides themselves. One exception is the recent approval of thesmall ω-conotoxin MVIIA peptide (Ziconotide™) for treatment ofintractable pain. The synthetic and refolding production process forZiconotide™, however, is costly and only results in a small peptideproduct with a very short half-life in vivo (about 4 hours).

A cost-effective process for producing therapeutics, such as but notlimited to, inhibitors of ion channels, is a desideratum provided by thepresent invention, which involves toxin peptides fused, or otherwisecovalently conjugated to a vehicle.

SUMMARY OF THE INVENTION

The present invention relates to a composition of matter of the formula:(X¹)_(a)—(F¹)_(d)—(X²)_(b)—(F²)_(e)—(X³)_(c)  (I)

and multimers thereof, wherein:

-   -   F¹ and F² are half-life extending moieties, and d and e are each        independently 0 or 1, provided that at least one of d and e is        1;    -   X¹, X², and X³ are each independently -(L)_(f)-P-(L)_(g)-, and f        and g are each independently 0 or 1;    -   P is a toxin peptide of no more than about 80 amino acid        residues in length, comprising at least two intrapeptide        disulfide bonds;    -   L is an optional linker (present when f=1 and/or g=1); and    -   a, b, and c are each independently 0 or 1, provided that at        least one of a, b and c is 1.    -   The present invention thus concerns molecules having variations        on Formula 1, such as the formulae:        P-(L)_(g)-F¹ (i.e., b, c, and e equal to 0);  (II)        F¹-(L)_(f)-P (i.e., a, c, and e equal to 0);  (III)        P-(L)_(g)-F¹-(L)_(f)-P or (X¹)_(a)—F¹—(X²)_(b) (i.e., c and e        equal to 0);  (IV)        F¹-(L)_(f)-P-(L)_(g)-F² (i.e., a and c equal to 0);  (V)        F¹-(L)_(f)-P-(L)_(g)-F²-(L)_(f)-P (i.e., a equal to 0);  (VI)        F¹—F²-(L)_(f)-P (i.e., a and b equal to 0);  (VII)        P-(L)_(g)-F¹—F² (i.e., b and c equal to 0);  (VIII)        P-(L)_(g)-F¹—F²-(L)_(f)-P (i.e., b equal to 0);  (IX)

and any multimers of any of these, when stated conventionally with theN-terminus of the peptide(s) on the left. All of such molecules ofFormulae II-IX are within the meaning of Structural Formula I. Withinthe meaning of Formula I, the toxin peptide (P), if more than one ispresent, can be independently the same or different from any other toxinpeptide(s) also present in the inventive composition, and the linkermoiety ((L)_(f) and/or (L)_(g)), if present, can be independently thesame or different from any other linker, or linkers, that may be presentin the inventive composition. Conjugation of the toxin peptide(s) to thehalf-life extending moiety, or moieties, can be via the N-terminaland/or C-terminal of the toxin peptide, or can be intercalary as to itsprimary amino acid sequence, F¹ being linked closer to the toxinpeptide's N-terminus than is linked F². Examples of useful half-lifeextending moieties (F¹ or F²) include immunoglobulin Fc domain, humanserum albumin (HSA), or poly(ethylene glycol) (PEG). These and otherhalf-life extending moieties described herein are useful, eitherindividually or in combination.

The present invention also relates to a composition of matter, whichincludes, conjugated or unconjugated, a toxin peptide analog of ShK,OSK1, ChTx, or Maurotoxin modified from the native sequences at one ormore amino acid residues, having greater Kv1.3 or IKCa1 antagonistactivity, and/or target selectivity, compared to a ShK, OSK1, orMaurotoxin (MTX) peptides having a native sequence. The toxin peptideanalogs comprise an amino acid sequence selected from any of thefollowing:

-   SEQ ID NOS: 88, 89, 92, 148 through 200, 548 through 561, 884    through 949, or 1295 through 1300 as set forth in Table 2; or-   SEQ ID NOS: 980 through 1274, 1303, or 1308 as set forth in Table 7;    or-   SEQ ID NOS: 330 through 337, 341, 1301, 1302, 1304 through 1307,    1309, 1311, 1312, and 1315 through 1336 as set forth in Table 13; or-   SEQ ID NOS: 36, 59, 344-346, or 1369 through 1390 as set forth in    Table 14.

The present invention also relates to other toxin peptide analogs thatcomprise an amino acid sequence selected from any of the following:

-   SEQ ID NOS: 201 through 225 as set forth in Table 3; or-   SEQ ID NOS: 242 through 248 or 250 through 260 as set forth in Table    4; or-   SEQ ID NOS: 261 through 275 as set forth in Table 5; or-   SEQ ID NOS: 276 through 293 as set forth in Table 6; or-   SEQ ID NOS: 299 through 315 as set forth in Table 8; or-   SEQ ID NOS: 316 through 318 as set forth in Table 9; or-   SEQ ID NO: 319 as set forth in Table 10; or-   SEQ ID NO: 327 or 328 as set forth in Table 11; or-   SEQ ID NOS: 330 through 337, 341, 1301, 1302, 1304 through 1307,    1309, 1311, 1312, or 1315 through 1336 as set forth in Table 13;-   SEQ ID NOS: 1369 through 1390 as set forth in Table 14; or-   SEQ ID NOS: 348 through 353 as set forth in Table 16; or-   SEQ ID NOS: 357 through 362, 364 through 368, 370, 372 through 385,    or 387 through 398 as set forth in Table 19; or-   SEQ ID NOS: 399 through 408 as set forth in Table 20; or-   SEQ ID NOS: 410 through 421 as set forth in Table 22; or-   SEQ ID NOS: 422, 424, 426, or 428 as set forth in Table 23; or-   SEQ ID NOS: 430 through 437 as set forth in Table 24; or-   SEQ ID NOS: 438 through 445 as set forth in Table 25; or-   SEQ ID NOS: 447, 449, 451, 453, 455, or 457 as set forth in Table    26; or-   SEQ ID NOS: 470 through 482 or 484 through 493 as set forth in Table    28; or-   SEQ ID NOS: 495 through 506 as set forth in Table 29; or-   SEQ ID NOS: 507 through 518 as set forth in Table 30.

The present invention is also directed to a pharmaceutical compositionthat includes the inventive composition of matter and a pharmaceuticallyacceptable carrier.

The compositions of this invention can be prepared by conventionalsynthetic methods, recombinant DNA techniques, or any other methods ofpreparing peptides and fusion proteins well known in the art.Compositions of this invention that have non-peptide portions can besynthesized by conventional organic chemistry reactions, in addition toconventional peptide chemistry reactions when applicable.

The primary use contemplated is as therapeutic and/or prophylacticagents. The inventive compositions incorporating the toxin peptide canhave activity and/or ion channel target selectivity comparable to—oreven greater than—the unconjugated peptide.

Accordingly, the present invention includes a method of treating anautoimmune disorder, which involves administering to a patient who hasbeen diagnosed with an autoimmune disorder, such as multiple sclerosis,type 1 diabetes, psoriasis, inflammatory bowel disease, contact-mediateddermatitis, rheumatoid arthritis, psoriatic arthritis, asthma, allergy,restinosis, systemic sclerosis, fibrosis, scleroderma,glomerulonephritis, Sjogren syndrome, inflammatory bone resorption,transplant rejection, graft-versus-host disease, or lupus, atherapeutically effective amount of the inventive composition of matter(preferably comprising a Kv1.3 antagonist peptide or IKCa1 antagonistpeptide), whereby at least one symptom of the disorder is alleviated inthe patient.

The present invention is further directed to a method of preventing ormitigating a relapse of a symptom of multiple sclerosis, which methodinvolves administering to a patient, who has previously experienced atleast one symptom of multiple sclerosis, a prophylactically effectiveamount of the inventive composition of matter (preferably comprising aKv1.3 antagonist peptide or IKCa1 antagonist peptide), such that the atleast one symptom of multiple sclerosis is prevented from recurring oris mitigated.

Although mostly contemplated as therapeutic agents, compositions of thisinvention can also be useful in screening for therapeutic or diagnosticagents. For example, one can use an Fc-peptide in an assay employinganti-Fc coated plates. The half-life extending moiety, such as Fc, canmake insoluble peptides soluble and thus useful in a number of assays.

Numerous additional aspects and advantages of the present invention willbecome apparent upon consideration of the figures and detaileddescription of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows schematic structures of some exemplary Fc dimers that canbe derived from an IgG1 antibody. “Fc” in the figure represents any ofthe Fc variants within the meaning of “Fc domain” herein. “X1” and “X2”represent peptides or linker-peptide combinations as definedhereinafter. The specific dimers are as follows:

FIG. 1A and FIG. 1D: Single disulfide-bonded dimers;

FIG. 1B and FIG. 1E: Doubly disulfide-bonded dimers;

FIG. 1C and FIG. 1F: Noncovalent dimers.

FIG. 2 shows schematic structures of some embodiments of the compositionof the invention that shows a single unit of the pharmacologicallyactive toxin peptide. FIG. 2A shows a single chain molecule and can alsorepresent the DNA construct for the molecule. FIG. 2B shows a dimer inwhich the linker-peptide portion is present on only one chain of thedimer. FIG. 2C shows a dimer having the peptide portion on both chains.The dimer of FIG. 2C will form spontaneously in certain host cells uponexpression of a DNA construct encoding the single chain shown in FIG.2A. In other host cells, the cells could be placed in conditionsfavoring formation of dimers or the dimers can be formed in vitro.

FIG. 3 shows exemplary nucleic acid and amino acid sequences (SEQ IDNOS: 1 and 2, respectively) of human IgG1 Fc that is optimized formammalian expression and can be used in this invention.

FIG. 4 shows exemplary nucleic acid and amino acid sequences (SEQ IDNOS: 3 and 4, respectively) of human IgG1 Fc that is optimized forbacterial expression and can be used in this invention.

FIG. 5A shows the amino acid sequence of the mature ShK peptide (SEQ IDNO: 5), which can be encoded for by a nucleic acid sequence containingcodons optimized for expression in mammalian cell, bacteria or yeast.

FIG. 5B shows the three disulfide bonds (—S—S—) formed by the sixcysteines within the ShK peptide (SEQ ID NO: 10).

FIG. 6 shows an alignment of the voltage-gated potassium channelinhibitor Stichodactyla helianthus (ShK) with other closely relatedmembers of the sea anemone toxin family. The sequence of the 35 aminoacid mature ShK toxin (Accession #P29187) isolated from the venom ofStichodactyla helianthus is shown aligned to other closely relatedmembers of the sea anemone family. The consensus sequence and predicteddisulfide linkages are shown, with highly conserved residues beingshaded. The HmK peptide toxin sequence shown (Swiss-Protein Accession#097436) is of the immature precursor from the Magnificent sea anemone(Radianthus magnifica; Heteractis magnifica) and the putative signalpeptide is underlined. The mature HmK peptide toxin would be predictedto be 35 amino acids in length and span residues 40 through 74. AeK isthe mature peptide toxin, isolated from the venom of the sea anemoneActinia equine (Accession #P81897). The sequence of the mature peptidetoxin AsKS (Accession #Q9TWG1) and BgK (Accession #P29186) isolated fromthe venom of the sea anemone Anemonia sulcata and Bunodosomagranulifera, respectively, are also shown. FIG. 6A shows the amino acidalignment (SEQ ID NO: 10) of ShK to other members of the sea anemonefamily of toxins, HmK (SEQ ID NO: 6 (Mature Peptide), (SEQ ID NO: 542,Signal and Mature Peptide portions)), AeK (SEQ ID NO: 7), AsKs (SEQ IDNO: 8), and BgK (SEQ ID NO: 9). The predicted disulfide linkages areshown and conserved residues are highlighted. (HmK, SEQ ID NO: 543; ShK,SEQ ID NO: 10; AeK, SEQ ID NO: 544; AsKS, SEQ ID NO: 545). FIG. 6B showsa disulfide linkage map for this family having 3 disulfide linkages(C1-C6, C2-C4, C3-C5).

FIG. 7 shows an amino acid alignment of the alpha-scorpion toxin familyof potassium channel inhibitors. (BmKK1, SEQ ID NO: 11; BmKK4, SEQ IDNO: 12; PBTx1, SEQ ID NO: 14; Tc32, SEQ ID NO: 13; BmKK6, SEQ ID NO: 15;P01, SEQ ID NO: 16; Pi2, SEQ ID NO: 17; Pi3, SEQ ID NO: 18; Pi4, SEQ IDNO: 19; MTX, SEQ ID NO: 20; Pi1, SEQ ID NO: 21; HsTx1, SEQ ID NO: 61;AgTx2, SEQ ID NO: 23; KTX1, SEQ ID NO: 24; OSK1, SEQ ID NO: 25; BmKTX,SEQ ID NO: 22; HgTX1, SEQ ID NO: 27; MgTx, SEQ ID NO: 28; C11Tx1, SEQ IDNO: 29; NTX, SEQ ID NO: 30; Tc30, SEQ ID NO: 31; TsTX-Ka, SEQ ID NO: 32;PBTx3, SEQ ID NO: 33; Lqh 15-1, SEQ ID NO: 34; MartenTx, SEQ ID NO: 37;ChTx, SEQ ID NO:36; ChTx-Lq2, SEQ ID NO: 42; IbTx, SEQ ID NO: 38; SloTx,SEQ ID NO: 39; BmTx1; SEQ ID NO: 43; BuTx, SEQ ID NO: 41; AmmTx3, SEQ IDNO: 44; AaTX1, SEQ ID NO: 45; BmTX3, SEQ ID NO: 46; Tc1, SEQ ID NO: 48;OSK2, SEQ ID NO: 49; TsK, SEQ ID NO: 54; CoTx1, SEQ ID NO:55; CoTx2, SEQID NO: 871; BmPo5, SEQ ID NO: 60; ScyTx, SEQ ID NO: 51; P05, SEQ ID NO:52; Tamapin, SEQ ID NO: 53; and TmTx, SEQ ID NO: 691. Highly conservedresidues are shaded and a consensus sequence is listed. Subfamilies ofthe α-KTx are listed and are from Rodriguez de la Vega, R. C. et al.(2003) TIPS 24: 222-227. A list of some ion channels reported toantagonized is listed (IK=IKCa, BK=BKCa, SK=SKCa, Kv=voltage-gated K+channels). Although most family members in this alignment represent themature peptide product, several represent immature or modified forms ofthe peptide and these include: BmKK1, BmKK4, BmKK6, BmKTX, MartenTx,ChTx, ChTx-Lq2, BmTx1, AaTx1, BmTX3, TsK, CoTx1, BmP05.

FIG. 8 shows an alignment of toxin peptides converted to peptibodies inthis invention (Apamin, SEQ ID NO: 68; HaTx1, SEQ ID NO: 494; ProTx1,SEQ ID NO: 56; PaTx2, SEQ ID NO: 57; ShK[2-35], SEQ ID NO: 92;ShK[1-35], SEQ ID NO: 5; HmK, SEQ ID NO: 6; ChTx (K32E), SEQ ID NO: 59;ChTx, SEQ ID NO: 36; IbTx, SEQ ID NO: 38; OSK1 (E16K, K20D), SEQ ID NO:296; OSK1, SEQ ID NO: 25; AgTx2, SEQ ID NO: 23; KTX1, SEQ ID NO: 24;MgTx, SEQ ID NO: 28; NTX, SEQ ID NO: 30; MTX, SEQ ID NO: 20; Pi2, SEQ IDNO: 17; HsTx1, SEQ ID NO: 61; Anuroctoxin [AnTx], SEQ ID NO: 62; BeKm1,SEQ ID NO: 63; ScyTx, SEQ ID NO: 51; ωGVIA, SEQ ID NO: 64; ωMVIIa, SEQID NO: 65; Ptu1, SEQ ID NO: 66; and CTX, SEQ ID NO: 67). The originalsources of the toxins is indicated, as well as, the number of cysteinesin each. Key ion channels targeted are listed. The alignment showsclustering of toxin peptides based on their source and ion channeltarget impact.

FIG. 9 shows disulfide arrangements within the toxin family. The numberof disulfides and the disulfide bonding order for each subfamily isindicated. A partial list of toxins that fall within each disulfidelinkage category is presented.

FIG. 10 illustrates that solution structures of toxins reveal a compactstructure. Solution structures of native toxins from sea anemone (ShK),scorpion (MgTx, MTX, HsTx1), marine cone snail (wGVIA) and tarantula(HaTx1) indicate the 28 to 39 amino acid peptides all form a compactstructure. The toxins shown have 3 or 4 disulfide linkages and fallwithin 4 of the 6 subfamilies shown in FIG. 9. The solution structuresof native toxins from sea anemone (ShK), scorpion (MgTx, MTX, HsTx1),marine cone snail (wGVIA) and tarantula (HaTx1) were derived fromProtein Data Bank (PDB) accession numbers 1ROO (mmdbld:5247), 1MTX(mmdbld:4064), 1TXM (mmdbld:6201), 1QUZ (mmdbld:36904), 1OMZ(mmdbld:1816) and 1D1H (mmdbld:14344) using the MMDB Entrez 3D-structuredatabase [J. Chen et al. (2003) Nucleic Acids Res. 31, 474] and viewer.

FIG. 11A-C shows the nucleic acid (SEQ ID NO: 69 and SEQ ID NO: 1358)and encoded amino acid (SEQ ID NO:70, SEQ ID NO:1359 and SEQ ID NO:1360) sequences of residues 5131-6660 of pAMG21ampR-Fc-pep. Thesequences of the Fc domain (SEQ ID NOS: 71 and 72) exclude the fiveC-terminal glycine residues. This vector enables production ofpeptibodies in which the peptide-linker portion is at the C-terminus ofthe Fc domain.

FIG. 11D shows a circle diagram of a peptibody bacterial expressionvector pAMG21ampR-Fc-pep having a chloramphenicol acetyltransferase gene(cat; “CmR” site) that is replaced with the peptide-linker sequence.

FIG. 12A-C shows the nucleic acid (SEQ ID NO: 73 and SEQ ID NO: 1361)and encoded amino acid (SEQ ID NO:74, SEQ ID NO: 1362 and SEQ ID NO:1363) sequences of residues 5131-6319 of pAMG21ampR-Pep-Fc. Thesequences of the Fc domain (SEQ ID NOS: 75 and 76) exclude the fiveN-terminal glycine residues. This vector enables production ofpeptibodies in which the peptide-linker portion is at the N-terminus ofthe Fc domain.

FIG. 12D shows a circle diagram of a peptibody bacterial expressionvector having a zeocin resistance (ble; “ZeoR”) site that is replacedwith the peptide-linker sequence.

FIG. 12E-G shows the nucleic acid (SEQ ID NO:1339) and encoded aminoacid sequences of pAMG21ampR-Pep-Fc (SEQ ID NO:1340, SEQ ID NO:1341, andSEQ ID NO:1342). The sequences of the Fc domain (SEQ ID NOS: 75 and 76)exclude the five N-terminal glycine residues. This vector enablesproduction of peptibodies in which the peptide-linker portion is at theN-terminus of the Fc domain.

FIG. 13A is a circle diagram of mammalian expression vector pCDNA3.1 (+)CMVi.

FIG. 13B is a circle diagram of mammalian expression vectorpCDNA3.1(+)CMVi-Fc-2xG4S-Activin Rllb that contains a Fc region fromhuman IgG1, a 10 amino acid linker and the activin Rllb gene.

FIG. 13C is a circle diagram of the CHO expression vector pDSRa22containing the Fc-L10-ShK[2-35] coding sequence.

FIG. 14 shows the nucleotide and encoded amino acid sequences (SEQ. ID.NOS: 77 and 78, respectively) of the molecule identified as“Fc-L10-ShK[1-35]” in Example 1 hereinafter. The L10 linker amino acidsequence (SEQ ID NO: 79) is underlined.

FIG. 15 shows the nucleotide and encoded amino acid sequences (SEQ. ID.NOS: 80 and 81, respectively) of the molecule identified as“Fc-L10-ShK[2-35]” in Example 2 hereinafter. The same L10 linker aminoacid sequence (SEQ ID NO: 79) as used in Fc-L10-ShK[1-35] (FIG. 14) isunderlined.

FIG. 16 shows the nucleotide and encoded amino acid sequences (SEQ. ID.NOS: 82 and 83, respectively) of the molecule identified as“Fc-L25-ShK[2-35]” in Example 2 hereinafter. The L25 linker amino acidsequence (SEQ ID NO: 84) is underlined.

FIG. 17 shows a scheme for N-terminal PEGylation of ShK peptide (SEQ IDNO: 5 and SEQ ID NO:10) by reductive amination, which is also describedin Example 32 hereinafter.

FIG. 18 shows a scheme for N-terminal PEGylation of ShK peptide (SEQ IDNO: 5 and SEQ ID NO:10) via amide formation, which is also described inExample 34 hereinafter.

FIG. 19 shows a scheme for N-terminal PEGylation of ShK peptide (SEQ IDNO: 5 and SEQ ID NO:10) by chemoselective oxime formation, which is alsodescribed in Example 33 hereinafter.

FIG. 20A shows a reversed-phase HPLC analysis at 214 nm and FIG. 20Bshows electrospray mass analysis of folded ShK[2-35], also described asfolded-“Des-Arg1-ShK” (Peptide 2).

FIG. 21 shows reversed phase HPLC analysis at 214 nm of N-terminallyPEGylated ShK[2-35], also referred to as N-TerminallyPEGylated-“Des-Arg1-ShK”.

FIG. 22A shows a reversed-phase HPLC analysis at 214 nm of foldedShK[1-35], also referred to as “ShK”.

FIG. 22B shows electrospray mass analysis of folded ShK[1-35], alsoreferred to as “ShK”.

FIG. 23 illustrates a scheme for N-terminal PEGylation of ShK[2-35] (SEQID NO: 92 and SEQ ID NO: 58, also referred to as “Des-Arg1-ShK” or “ShKd1”) by reductive amination, which is also described in Example 31hereinafter.

FIG. 24A shows a western blot of conditioned medium from HEK 293 cellstransiently transfected with Fc-L10-ShK[1-35]. Lane 1: molecular weightmarkers; Lane 2: 15 μl Fc-L10-ShK;

Lane 3: 10 μl Fc-L10-ShK; Lane 4: 5 μl Fc-L10-ShK; Lane 5; molecularweight markers; Lane 6: blank; Lane 7: 15 μl No DNA control; Lane 8: 10μl No DNA control; Lane 9: 5 μl No DNA control; Lane 10; molecularweight markers.

FIG. 24B shows a western blot of with 15 μl of conditioned medium fromclones of Chinese Hamster Ovary (CHO) cells stably transfected withFc-L-ShK[1-35]. Lanes 1-15 were loaded as follows: blank, BB6, molecularweight markers, BB5, BB4, BB3, BB2, BB1, blank, BD6, BD5, molecularweight markers, BD4, BD3, BD2.

FIG. 25A shows a western blot of a non-reducing SDS-PAGE gel containingconditioned medium from 293T cells transiently transfected withFc-L-SmIIIA.

FIG. 25B shows a western blot of a reducing SDS-PAGE gel containingconditioned medium from 293T cells transiently transfected withFc-L-SmIIIA.

FIG. 26A shows a Spectral scan of 10 μl purified Fc-L10-ShK[1-35]product from stably transfected CHO cells diluted in 700 μl PBS(blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a1-cm path length quartz cuvette.

FIG. 26B shows Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE of the final Fc-L10-ShK[1-35] product. Lanes 1-12 were loadedas follows: Novex Mark 12 wide range protein standards, 0.5 μg productnon-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg productnon-reduced, Novex Mark 12 wide range protein standards, 0.5 μg productreduced, blank, 2.0 μg product reduced, blank, and 10 μg productreduced.

FIG. 26C shows size exclusion chromatography on 20 μg of the finalFc-L10-ShK[1-35] product injected on to a Phenomenex BioSep SEC 3000column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, and pH 6.9 at 1ml/min observing the absorbance at 280 nm.

FIG. 26D shows a MALDI mass spectral analysis of the final sample ofFc-L10-ShK[1-35] analyzed using a Voyager DE-RP time-of-flight massspectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). Thepositive ion/linear mode was used, with an accelerating voltage of 25kV. Each spectrum was produced by accumulating data from ˜200 lasershots. External mass calibration was accomplished using purifiedproteins of known molecular masses.

FIG. 27A shows a Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE of the final purified Fc-L10-ShK[2-35] product from stablytransfected CHO cells. Lane 1-12 were loaded as follows: Novex Mark 12wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μgproduct non-reduced, blank, 10 μg product non-reduced, Novex Mark 12wide range protein standards, 0.5 μg product reduced, blank, 2.0 μgproduct reduced, blank, and 10 μg product reduced.

FIG. 27B shows size exclusion chromatography on 50 μg of the purifiedFc-L10-ShK[2-35] injected on to a Phenomenex BioSep SEC 3000 column(7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, and pH 6.9 at 1 ml/minobserving the absorbance at 280 nm.

FIG. 27C shows a Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE of Fc-L5-ShK[2-35] purified from stably transfected CHO cells.Lane 1-12 are loaded as follows: Novex Mark 12 wide range proteinstandards, 0.5 μg product non-reduced, blank, 2.0 μg productnon-reduced, blank, 10 μg product non-reduced, Novex Mark 12 wide rangeprotein standards, 0.5 μg product reduced, blank, 2.0 μg productreduced, blank, and 10 μg product reduced.

FIG. 27D shows a Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE of Fc-L25-ShK[2-35] purified from stably transfected CHO cells.Lane 1-12 are loaded as follows: Novex Mark 12 wide range proteinstandards, 0.5 μg product non-reduced, blank, 2.0 μg productnon-reduced, blank, 10 μg product non-reduced, Novex Mark 12 wide rangeprotein standards, 0.5 μg product reduced, blank, 2.0 μg productreduced, blank, and 10 μg product reduced.

FIG. 27E shows a spectral scan of 10 μl of the Fc-L10-ShK[2-35] productdiluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 27F shows a MALDI mass spectral analysis of the final sample ofFc-L10-ShK[2-35] analyzed using a Voyager DE-RP time-of-flight massspectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). Thepositive ion/linear mode was used, with an accelerating voltage of 25kV. Each spectrum was produced by accumulating data from about 200 lasershots. External mass calibration was accomplished using purifiedproteins of known molecular masses.

FIG. 27G shows a spectral scan of 10 μl of the Fc-L5-ShK[2-35] productdiluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 27H shows the size exclusion chromatography on 50 mg of the finalFc-L5-ShK[2-35] product injected on to a Phenomenex BioSep SEC 3000column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/minobserving the absorbance at 280 nm.

FIG. 27I shows a MALDI mass spectral analysis of the final sample ofFc-L5-ShK[2-35] analyzed using a Voyager DE-RP time-of-flight massspectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). Thepositive ion/linear mode was used, with an accelerating voltage of 25kV. Each spectrum was produced by accumulating data from ˜200 lasershots. External mass calibration was accomplished using purifiedproteins of known molecular masses.

FIG. 27J shows a Spectral scan of 20 μl of the product diluted in 700 μlPBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer anda 1 cm path length quartz cuvette.

FIG. 27K shows the size exclusion chromatography on 50 μg of the finalFc-L25-ShK[2-35] product injected on to a Phenomenex BioSep SEC 3000column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/minobserving the absorbance at 280 nm.

FIG. 27L shows a MALDI mass spectral analysis of the final sample ofFc-L25-ShK[2-35] analyzed using a Voyager DE-RP time-of-flight massspectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). Thepositive ion/linear mode was used, with an accelerating voltage of 25kV. Each spectrum was produced by accumulating data from about 200 lasershots. External mass calibration was accomplished using purifiedproteins of known molecular masses.

FIG. 28A shows a Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE of Fc-L10-KTX1 purified and refolded from bacterial cells. Lane1-12 are loaded as follows: Novex Mark 12 wide range protein standards,0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10μg product non-reduced, Novex Mark 12 wide range protein standards, 0.5μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μgproduct reduced.

FIG. 28B shows size exclusion chromatography on 45 μg of purifiedFc-L10-KTX1 injected on to a Phenomenex BioSep SEC 3000 column (7.8×300mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observing theabsorbance at 280 nm.

FIG. 28C shows a Spectral scan of 20 μl of the Fc-L10-KTX1 productdiluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 28D shows a MALDI mass spectral analysis of the final sample ofFc-L10-KTX1 analyzed using a Voyager DE-RP time-of-flight massspectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). Thepositive ion/linear mode was used, with an accelerating voltage of 25kV. Each spectrum was produced by accumulating data from ˜200 lasershots. External mass calibration was accomplished using purifiedproteins of known molecular masses.

FIG. 29A shows a Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE of Fc-L-AgTx2 purified and refolded from bacterial cells. Lane1-12 are loaded as follows: Novex Mark 12 wide range protein standards,0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10μg product non-reduced, Novex Mark 12 wide range protein standards, 0.5μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μgproduct reduced.

FIG. 29B shows a Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE of Fc-L10-HaTx1 purified and refolded from bacterial cells.Lane 1-12 are loaded as follows: Novex Mark 12 wide range proteinstandards, 0.5 μg product non-reduced, blank, 2.0 μg productnon-reduced, blank, 10 μg product non-reduced, Novex Mark 12 wide rangeprotein standards, 0.5 μg product reduced, blank, 2.0 μg productreduced, blank, and 10 μg product reduced, spectral scan of the purifiedmaterial.

FIG. 29C shows a Spectral scan of 20 μl of the Fc-L10-AgTx2 productdiluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 29D shows the Size exclusion chromatography on 20 μg of the finalFc-L10-AgTx2 product injected on to a Phenomenex BioSep SEC 3000 column(7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observingthe absorbance at 280 nm.

FIG. 29E shows a MALDI mass spectral analysis of the final sample ofFc-L10-AgTx2 analyzed using a Voyager DE-RP time-of-flight massspectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). Thepositive ion/linear mode was used, with an accelerating voltage of 25kV. Each spectrum was produced by accumulating data from about 200 lasershots. External mass calibration was accomplished using purifiedproteins of known molecular masses.

FIG. 29F shows a Spectral scan of 20 μl of the Fc-L10-HaTx1 productdiluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 29G shows the size exclusion chromatography on 20 μg of the finalFc-L10-HaTx1 product injected on to a Phenomenex BioSep SEC 3000 column(7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observingthe absorbance at 280 nm.

FIG. 29H shows a MALDI mass spectral analysis of the final sample ofFc-L10-HaTx1 analyzed using a Voyager DE-RP time-of-flight massspectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). Thepositive ion/linear mode was used, with an accelerating voltage of 25kV. Each spectrum was produced by accumulating data from ˜200 lasershots. External mass calibration was accomplished using purifiedproteins of known molecular masses.

FIG. 30A shows Fc-L10-ShK[1-35] purified from CHO cells produces aconcentration dependent block of the outward potassium current recordedfrom HEK293 cell stably expressing the human Kv1.3 channel.

FIG. 30B shows the time course of potassium current block byFc-L10-ShK[1-35] at various concentrations. The IC50 was estimated to be15±2 pM (n=4 cells).

FIG. 30C shows synthetic ShK[1-35] (also referred to as “ShK” alone)produces a concentration dependent block of the outward potassiumcurrent recorded from HEK293 cell stably expressing human Kv1.3 channel.

FIG. 30D shows the time course of ShK[1-35] block at variousconcentrations. The IC50 for ShK was estimated to be 12±1 pM (n=4cells).

FIG. 31A shows synthetic peptide analog ShK[2-35] producing aconcentration dependent block of the outward potassium current asrecorded from HEK293 cells stably expressing human Kv1.3 channel with anIC50 of 49±5 pM (n=3 cells).

FIG. 31B shows the CHO-derived Fc-L10-ShK[2-35] peptibody producing aconcentration dependent block of the outward potassium current asrecorded from HEK293 cell stably expressing human Kv1.3 channel with anIC50 of 115±18 pM (n=3 cells).

FIG. 31C shows the Fc-L5-ShK[2-35] peptibody produces a concentrationdependent block of the outward potassium current recorded from HEK293cell stably expressing human Kv1.3 channel with an IC50 of 100 pM (n=3cells).

FIG. 32A shows Fc-L-KTX1 peptibody purified from bacterial cellsproducing a concentration dependent block of the outward potassiumcurrent as recorded from HEK293 cell stably expressing human Kv1.3channel.

FIG. 32B shows the time course of potassium current block by Fc-L10-KTX1at various concentrations.

FIG. 33 shows by immunohistochemistry that CHO-derived Fc-L10-ShK[1-35]peptibody stains HEK 293 cells stably transfected with human Kv1.3 (FIG.33A), whereas untransfected HEK 293 cells are not stained with thepeptibody (FIG. 33B).

FIG. 34 shows results of an enzyme-immunoassay using fixed HEK 293 cellsstably transfected with human Kv1.3. FIG. 34A shows the CHO-derivedFc-L10-ShK[1-35] (referred to here simply as “Fc-L10-ShK”) peptibodyshows a dose-dependent increase in response, whereas the CHO-Fc control(“Fc control”) does not. FIG. 34B shows the Fc-L10-ShK[1-35] peptibody(referred to here as “Fc-ShK”) does not elicit a response fromuntransfected HEK 293 cells using similar conditions and also showsother negative controls.

FIG. 35 shows the CHO-derived Fc-L10-ShK[1-35] peptibody shows adose-dependent inhibition of IL-2 (FIG. 35A) and IFNγ (FIG. 35B)production from PMA and αCD3 antibody stimulated human PBMCs. Thepeptibody shows a novel pharmacology exhibiting a complete inhibition ofthe response, whereas the synthetic ShK[1-35] peptide alone shows only apartial inhibition.

FIG. 36 shows the mammalian-derived Fc-L10-ShK[1-35] peptibody inhibitsT cell proliferation (³H-thymidine incorporation) in human PBMCs fromtwo normal donors stimulated with antibodies to CD3 and CD28. FIG. 36Ashows the response of donor 1 and FIG. 36B the response of donor 2.Pre-incubation with the anti-CD32 (FcgRII) blocking antibody did notalter the sensitivity toward the peptibody.

FIG. 37 shows the purified CHO-derived Fc-L10-ShK[1-35] peptibody causesa dose-dependent inhibition of IL-2 (FIG. 37A) and IFNγ (FIG. 37B)production from αCD3 and αCD28 antibody stimulated human PBMCs.

FIG. 38A shows the PEGylated ShK[2-35] synthetic peptide produces aconcentration dependent block of the outward potassium current recordedfrom HEK293 cell stably expressing human Kv1.3 channel and the timecourse of potassium current block at various concentrations is shown inFIG. 38B.

FIG. 39A shows a spectral scan of 50 μl of the Fc-L10-ShK(1-35) productdiluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 39B shows a Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE of the final Fc-L10-ShK(1-35) product. Lane 1-12 are loaded asfollows: Novex Mark 12 wide range protein standards, 0.5 μg productnon-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg productnon-reduced, Novex Mark 12 wide range protein standards, 0.5 μg productreduced, blank, 2.0 μg product reduced, blank, and 10 μg productreduced.

FIG. 39C shows the Size exclusion chromatography on 50 μg of the finalFc-L10-ShK(1-35) product injected on to a Phenomenex BioSep SEC 3000column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/minobserving the absorbance at 280 nm.

FIG. 40A shows a Spectral scan of 20 μl of the Fc-L10-ShK(2-35) productdiluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 40B shows a Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE of the final Fc-L10-ShK(2-35) product. Lanes 1-12 are loaded asfollows: Novex Mark 12 wide range protein standards, 0.5 μg productnon-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg productnon-reduced, Novex Mark 12 wide range protein standards, 0.5 μg productreduced, blank, 2.0 μg product reduced, blank, and 10 μg productreduced.

FIG. 40C shows the size exclusion chromatography on 50 μg of the finalFc-L10-ShK(2-35) product injected on to a Phenomenex BioSep SEC 3000column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/minobserving the absorbance at 280 nm.

FIG. 40D shows a MALDI mass spectral analysis of the final sample ofFc-L10-ShK(2-35) analyzed using a Voyager DE-RP time-of-flight massspectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). Thepositive ion/linear mode was used, with an accelerating voltage of 25kV. Each spectrum was produced by accumulating data from ˜200 lasershots. External mass calibration was accomplished using purifiedproteins of known molecular masses.

FIG. 41A shows spectral scan of 50 μl of the Fc-L10-OSK1 product dilutedin 700 μl Formulation Buffer using a Hewlett Packard 8453spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 41B shows Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE of the final Fc-L10-OSK1 product. Lanes 1-12 are loaded asfollows: Novex Mark 12 wide range protein standards, 0.5 μg productnon-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg productnon-reduced, Novex Mark 12 wide range protein standards, 0.5 μg productreduced, blank, 2.0 μg product reduced, blank, and 10 μg productreduced.

FIG. 41C shows size exclusion chromatography on 123 μg of the finalFc-L10-OSK1 product injected on to a Phenomenex BioSep SEC 3000 column(7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observingthe absorbance at 280 nm.

FIG. 41D shows liquid chromatography—mass spectral analysis ofapproximately 4 μg of the final Fc-L10-OSK1 sample using a Vydac C₄column with part of the effluent directed into a LCQ ion trap massspectrometer. The mass spectrum was deconvoluted using the Bioworkssoftware provided by the mass spectrometer manufacturer.

FIG. 42A-B shows nucleotide and amino acid sequences (SEQ ID NO: 1040and SEQ ID NO: 1041, respectively) of Fc-L10-OSK1.

FIG. 43A-B shows nucleotide and amino acid sequences (SEQ ID NO: 1042and SEQ ID NO: 1043, respectively) of Fc-L10-OSK1[K7S].

FIG. 44A-B shows nucleotide and amino acid sequences (SEQ ID NO: 1044and SEQ ID NO: 1045, respectively) of Fc-L10-OSK1[E16K,K20D].

FIG. 45A-B shows nucleotide and amino acid sequences (SEQ ID NO: 1046and SEQ ID NO: 1047, respectively) of Fc-L10-OSK1[K7S,E16K,K20D].

FIG. 46 shows a Western blot (from tris-glycine 4-20% SDS-PAGE) withanti-human Fc antibodies. Lanes 1-6 were loaded as follows: 15 μl ofFc-L10-OSK1[K7S,E16K,K20D]; 15 μl of Fc-L10-OSK1[E16K,K20D]; 15 μl ofFc-L10-OSK1[K7S]; 15 μl of Fc-L10-OSK1; 15 μl of “No DNA” control;molecular weight markers

FIG. 47 shows a Western blot (from tris-glycine 4-20% SDS-PAGE) withanti-human Fc antibodies. Lanes 1-5 were loaded as follows: 2 μl ofFc-L10-OSK1; 5 μl of Fc-L10-OSK1; 10 μg of Fc-L10-OSK1; 20 ng Human IgGstandard; molecular weight markers.

FIG. 48 shows a Western blot (from tris-glycine 4-20% SDS-PAGE) withanti-human Fc antibodies. Lanes 1-13 were loaded as follows: 20 ng HumanIgG standard; D1; C3; C2; B6; B5; B2; B1; A6; A5; A4; A3; A2 (5 μl ofclone-conditioned medium loaded in lanes 2-13).

FIG. 49A shows a spectral scan of 50 μl of the Fc-L10-OsK1 productdiluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 49B shows Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE of the final Fc-L10-OsK1 product. Lane 1-12 are loaded asfollows: Novex Mark 12 wide range protein standards, 0.5 μg productnon-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg productnon-reduced, Novex Mark 12 wide range protein standards, 0.5 μg productreduced, blank, 2.0 μg product reduced, blank, and 10 μg productreduced.

FIG. 49C shows Size exclusion chromatography on 149 μg of the finalFc-L10-OsK1 product injected on to a Phenomenex BioSep SEC 3000 column(7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observingthe absorbance at 280 nm.

FIG. 49D shows MALDI mass spectral analysis of the final sample ofFc-L10-OsK1 analyzed using a Voyager DE-RP time-of-flight massspectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). Thepositive ion/linear mode was used, with an accelerating voltage of 25kV. Each spectrum was produced by accumulating data from ˜200 lasershots. External mass calibration was accomplished using purifiedproteins of known molecular masses.

FIG. 50A shows a spectral scan of 50 μl of the Fc-L10-OsK1(K7S) productdiluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 50B shows Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE of the final Fc-L10-OsK1(K7S) product. Lane 1-12 are loaded asfollows: Novex Mark 12 wide range protein standards, 0.5 μg productnon-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg productnon-reduced, Novex Mark 12 wide range protein standards, 0.5 μg productreduced, blank, 2.0 μg product reduced, blank, and 10 μg productreduced.

FIG. 50C shows size exclusion chromatography on 50 μg of the finalFc-L10-OsK1(K7S) product injected on to a Phenomenex BioSep SEC 3000column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/minobserving the absorbance at 280 nm.

FIG. 50D shows MALDI mass spectral analysis of a sample of the finalproduct Fc-L10-OsK1(K7S) analyzed using a Voyager DE-RP time-of-flightmass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse).The positive ion/linear mode was used, with an accelerating voltage of25 kV. Each spectrum was produced by accumulating data from ˜200 lasershots. External mass calibration was accomplished using purifiedproteins of known molecular masses.

FIG. 51A shows a spectral scan of 50 μl of the Fc-L10-OsK1(E16K, K20D)product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard8453 spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 51B shows Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE of the final Fc-L10-OsK1(E16K, K20D) product. Lane 1-12 areloaded as follows: Novex Mark 12 wide range protein standards, 0.5 μgproduct non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μgproduct non-reduced, Novex Mark 12 wide range protein standards, 0.5 μgproduct reduced, blank, 2.0 μg product reduced, blank, and 10 μg productreduced.

FIG. 51C shows size exclusion chromatography on 50 μg of the finalFc-L10-OsK1(E16K, K20D) product injected on to a Phenomenex BioSep SEC3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1ml/min observing the absorbance at 280 nm.

FIG. 51D shows MALDI mass spectral analysis of a sample of the finalproduct Fc-L10-OsK1(E16K, K20D) analyzed using a Voyager DE-RPtime-of-flight mass spectrometer equipped with a nitrogen laser (337 nm,3 ns pulse). The positive ion/linear mode was used, with an acceleratingvoltage of 25 kV. Each spectrum was produced by accumulating data from˜200 laser shots. External mass calibration was accomplished usingpurified proteins of known molecular masses.

FIG. 52A shows a spectral scan of 50 μl of the Fc-L10-OsK1(K7S, E16K,K20D) product diluted in 700 μl PBS (blanking buffer) using a HewlettPackard 8453 spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 52B shows Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE of the final Fc-L10-OsK1(K7S, E16K, K20D) product. Lanes 1-12are loaded as follows: Novex Mark 12 wide range protein standards, 0.5μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μgproduct non-reduced, Novex Mark 12 wide range protein standards, 0.5 μgproduct reduced, blank, 2.0 μg product reduced, blank, and 10 μg productreduced.

FIG. 52C shows size exclusion chromatography on 50 μg of the finalFc-L10-OsK1(K7S, E16K, K20D) product injected on to a Phenomenex BioSepSEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1ml/min observing the absorbance at 280 nm.

FIG. 52D shows MALDI mass spectral analysis of a sample of the finalproduct Fc-L10-OsK1(K7S, E16K, K20D) analyzed using a Voyager DE-RPtime-of-flight mass spectrometer equipped with a nitrogen laser (337 nm,3 ns pulse). The positive ion/linear mode was used, with an acceleratingvoltage of 25 kV. Each spectrum was produced by accumulating data from˜200 laser shots. External mass calibration was accomplished usingpurified proteins of known molecular masses.

FIG. 53 shows inhibition of the outward potassium current recorded fromHEK293 cell stably expressing human Kv1.3 channel by synthetic Osk1, a38-residue toxin peptide of the Asian scorpion Orthochirus scrobiculosusvenom. FIG. 53A shows a concentration dependent block of the outwardpotassium current recorded from HEK293 cell stably expressing humanKv1.3 channel by the synthetic Osk1 toxin peptide. FIG. 53B shows thetime course of the synthetic Osk1 toxin peptide block at variousconcentrations. The IC50 for the synthetic Osk1 toxin peptide wasestimated to be 39±12 pM (n=4 cells).

FIG. 54 shows that modification of the synthetic OSK1 toxin peptide byfusion to the Fc-fragment of an antibody (OSK1-peptibody) retained theinhibitory activity against the human Kv1.3 channel. FIG. 54A shows aconcentration dependent block of the outward potassium current recordedfrom HEK293 cells stably expressing human Kv1.3 channel by OSK1 linkedto a human IgG1 Fc-fragment with a linker chain length of 10 amino acidresidues (Fc-L10-OSK1). The fusion construct was stably expressed inChinese Hamster Ovarian (CHO) cells. FIG. 54B shows the time course ofthe Fc-L10-OSK1 block at various concentrations. The IC50 forFc-L10-OSK1 was estimated to be 198±35 pM (n=6 cells), approximately5-fold less potent than the synthetic OSK1 toxin peptide.

FIG. 55 shows that a single amino-acid residue substitution of theOSK1-peptibody retained the inhibitory activity against the human Kv1.3channel. FIG. 55A shows a concentration dependent block of the outwardpotassium current recorded from HEK293 cell stably expressing humanKv1.3 channel by OSK1-peptibody with a single amino acid substitution(lysine to serine at the 7^(th) position from N-terminal, [K7S]) andlinked to a human IgG1 Fc-fragment with a linker chain length of 10amino acid residues (Fc-L10-OSK1[K7S]). The fusion construct was stablyexpressed in Chinese Hamster Ovarian (CHO) cells. FIG. 55B shows thetime course of potassium current block by Fc-L10-OSK1[K7S] at variousconcentrations. The IC50 was estimated to be 372±71 pM (n=4 cells),approximately 10-fold less potent than the synthetic OSK1 toxin peptide.

FIG. 56 shows that a two amino-acid residue substitution of theOSK1-peptibody retained the inhibitory activity against the human Kv1.3channel. FIG. 56A shows a concentration dependent block of the outwardpotassium current recorded from HEK293 cell stably expressing humanKv1.3 channel by OSK1-peptibody with two amino acid substitutions(glutamic acid to lysine and lysine to aspartic acid at the 16^(th) and20^(th) position from N-terminal respectively, [E16KK20D]) and linked toa human IgG1 Fc-fragment with a linker chain length of 10 amino acidresidues (Fc-L10-OSK1[E16KK20D]). The fusion construct was stablyexpressed in Chinese Hamster Ovarian (CHO) cells. FIG. 56B shows thetime course of potassium current block by Fc-L10-OSK1[E16KK20D] atvarious concentrations. The IC50 was estimated to be 248±63 pM (n=3cells), approximately 6-fold less potent than the synthetic OSK1 toxinpeptide.

FIG. 57 shows that a triple amino-acid residue substitution of theOSK1-peptibody retained the inhibitory activity against the human Kv1.3channel, but the potency of inhibition was significantly reduced whencompared to the synthetic OSK1 toxin peptide. FIG. 57A shows aconcentration dependent block of the outward potassium current recordedfrom HEK293 cell stably expressing human Kv1.3 channel by OSK1-peptibodywith triple amino acid substitutions (lysine to serine, glutamic acid tolysine and lysine to aspartic acid at the 7^(th), 16^(th) and 20^(th)position from N-terminal respectively, [K7SE16KK20D]) and linked to ahuman IgG1 Fc-fragment with a linker chain length of 10 amino acidresidues (Fc-L10-OSK1[K7SE16KK20D]). The fusion construct was stablyexpressed in Chinese Hamster Ovarian (CHO) cells. FIG. 57B shows thetime course of potassium current block by Fc-L10-OSK1[K7SE16KK20D] atvarious concentrations. The IC50 was estimated to be 812±84 pM (n=3cells), approximately 21-fold less potent than the synthetic OSK1 toxinpeptide.

FIG. 58 shows Standard curves for ShK (FIG. 58A) and 20K PEG-ShK[1-35](FIG. 58B) containing linear regression equations for each Standard at agiven percentage of serum.

FIG. 59 shows the pharmacokinetic profile in rats of 20K PEG ShK[1-35]molecule after IV injection.

FIG. 60 shows Kv1.3 inhibitory activity in serum samples (5%) of ratsreceiving a single equal molar IV injection of Kv1.3 inhibitors ShKversus 20K PEG-ShK[1-35].

FIG. 61 illustrates an Adoptive Transfer EAE model experimental design(n=5 rats per treatment group). Dosing values in microgram per kilogram(mg/kg) are based on peptide content.

FIG. 62 shows that treatment with PEG-ShK ameliorated disease in rats inthe adoptive transfer EAE model. Clinical scoring: 0=No signs,0.5=distal limp tail, 1.0=limp tail, 2.0=mild paraparesis, ataxia,3.0=moderate paraparesis, 3.5=one hind leg paralysis, 4.0=complete hindleg paralysis, 5.0=complete hind leg paralysis and incontinence,5.5=tetraplegia, 6.0=moribund state or death. Rats reaching a score of5.5 to 6 died or were euthanized. Mean±sem values are shown. (n=5 ratsper treatment group.)

FIG. 63 shows that treatment with PEG-ShK prevented loss of body weightin the adoptive transfer EAE model. Rats were weighed on days—1, 4, 6,and 8 (for surviving rats). Mean±sem values are shown.

FIG. 64 shows that thapsigargin-induced IL-2 production in human wholeblood was suppressed by the Kv1.3 channel inhibitors ShK[1-35] andFc-L10-ShK[2-35]. The calcineurin inhibitor cyclosporine A also blockedthe response. The BKCa channel inhibitor iberiotoxin (IbTx) showed nosignificant activity. The response of whole blood from two separatedonors is shown in FIG. 64A and FIG. 64B.

FIG. 65 shows that thapsigargin-induced IFN-g production in human wholeblood was suppressed by the Kv1.3 channel inhibitors ShK[1-35] andFc-L10-ShK[2-35]. The calcineurin inhibitor cyclosporine A also blockedthe response. The BKCa channel inhibitor iberiotoxin (IbTx) showed nosignificant activity. The response of whole blood from two separatedonors is shown in FIG. 65A and FIG. 65B.

FIG. 66 shows that thapsigargin-induced upregulation of CD40L on T cellsin human whole blood was suppressed by the Kv1.3 channel inhibitorsShK[1-35] and Fc-L10-ShK[1-35] (Fc-ShK). The calcineurin inhibitorcyclosporine A (CsA) also blocked the response. FIG. 66A shows resultsof an experiment looking at the response of total CD4+ T cells. FIG. 66Bshows results of an experiment that looked at total CD4+ T cells, aswell as CD4+CD45+ and CD4+CD45− T cells. In FIG. 66B, the BKCa channelinhibitor iberiotoxin (IbTx) and the Kv1.1 channel inhibitordendrotoxin-K (DTX-K) showed no significant activity.

FIG. 67 shows that thapsigargin-induced upregulation of the IL-2R on Tcells in human whole blood was suppressed by the Kv1.3 channelinhibitors ShK[1-35] and Fc-L10-ShK[1-35] (Fc-ShK). The calcineurininhibitor cyclosporine A (CsA) also blocked the response. FIG. 67A showsresults of an experiment looking at the response of total CD4+ T cells.FIG. 67B shows results of an experiment that looked at total CD4+ Tcells, as well as CD4+CD45+ and CD4+CD45− T cells. In FIG. 67B, the BKCachannel inhibitor iberiotoxin (IbTx) and the Kv1.1 channel inhibitordendrotoxin-K (DTX-K) showed no significant activity.

FIG. 68 shows cation exchange chromatograms of PEG-peptide purificationon SP Sepharose HP columns for PEG-Shk purification (FIG. 68A) andPEG-OSK-1 purification (FIG. 68B).

FIG. 69 shows RP-HPLC chromatograms on final PEG-peptide pools todemonstrate purity of PEG-Shk purity>99% (FIG. 69A) and PEG-Osk1purity>97% (FIG. 69B).

FIG. 70 shows the amino acid sequence (SEQ ID NO: 976) of an exemplaryFcLoop-L2-OsK1-L2 having three linked domains: Fc N-terminal domain(amino acid residues 1-139); OsK1 (underlined amino acid residues142-179); and Fc C-terminal domain (amino acid residues 182-270).

FIG. 71 shows the amino acid sequence (SEQ ID NO: 977) of an exemplaryFcLoop-L2-ShK-L2 having three linked domains: Fc N-terminal domain(amino acid residues 1-139); ShK (underlined amino acid residues142-176); and Fc C-terminal domain (amino acid residues 179-267).

FIG. 72 shows the amino acid sequence (SEQ ID NO: 978) of an exemplaryFcLoop-L2-ShK-L4 having three linked domains: Fc N-terminal domain(amino acid residues 1-139); ShK (underlined amino acid residues142-176); and Fc C-terminal domain (amino acid residues 181-269).

FIG. 73 shows the amino acid sequence (SEQ ID NO: 979) of an exemplaryFcLoop-L4-OsK1-L2 having three linked domains: Fc N-terminal domain(amino acid residues 1-139); OsK1 (underlined amino acid residues144-181); and Fc C-terminal domain (amino acid residues 184-272).

FIG. 74 shows that the 20K PEGylated ShK[1-35] provided potent blockadeof human Kv1.3 as determined by whole cell patch clamp electrophysiologyon HEK293/Kv1.3 cells. The data represents blockade of peak current.

FIG. 75 shows schematic structures of some other exemplary embodimentsof the composition of matter of the invention. “X²” and “X³” representtoxin peptides or linker-toxin peptide combinations (i.e.,-(L)_(f)-P-(L)_(g)-) as defined herein. As described herein but notshown in FIG. 75, an additional X¹ domain and one or more additional PEGmoieties are also encompassed in. other embodiments. The specificembodiments shown here are as follows:

FIG. 75C, FIG. 75D, FIG. 75G and FIG. 75H: show a single chain moleculeand can also represent the DNA construct for the molecule.

FIG. 75A, FIG. 75B, FIG. 75E and FIG. 75F: show doubly disulfide-bondedFc dimers (in position F²); FIG. 75A and FIG. 75B show a dimer havingthe toxin peptide portion on both chains in position X³; FIG. 75E andFIG. 75F show a dimer having the toxin peptide portion on both chains Inposition X².

FIG. 76A shows a spectral scan of 50 μl of the ShK[2-35]-Fc productdiluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 76B shows Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE of the final ShK[2-35]-Fc product. Lanes 1-12 were loaded asfollows: Novex Mark 12 wide range protein standards, 0.5 μg productnon-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg productnon-reduced, Novex Mark 12 wide range protein standards, 0.5 μg productreduced, blank, 2.0 μg product reduced, blank, and 10 μg productreduced.

FIG. 76C shows size exclusion chromatography on 70 μg of the finalShK[2-35]-Fc product injected on to a Phenomenex BioSep SEC 3000 column(7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observingthe absorbance at 280 nm.

FIG. 76D shows LC-MS analysis of the final ShK[2-35]-Fc sample using anAgilent 1100 HPCL running reverse phase chromatography, with the columneffluent directly coupled to an electrospray source of a Thermo FinniganLCQ ion trap mass spectrometer. Relevant spectra were summed anddeconvoluted to mass data with the Bioworks software package.

FIG. 77A shows a spectral scan of 20 μl of the met-ShK[1-35]-Fc productdiluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 77B shows Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE of the final met-ShK[1-35]-Fc product. Lanes 1-12 were loadedas follows: Novex Mark 12 wide range protein standards, 0.5 μg productnon-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg productnon-reduced, Novex Mark 12 wide range protein standards, 0.5 μg productreduced, blank, 2.0 μg product reduced, blank, and 10 μg productreduced.

FIG. 77C shows size exclusion chromatography on 93 μg of the finalmet-ShK[1-35]-Fc product injected on to a Phenomenex BioSep SEC 3000column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/minobserving the absorbance at 280 nm.

FIG. 77D shows MALDI mass spectral analysis of the finalmet-ShK[1-35]-Fc sample analyzed using a Voyager DE-RP time-of-flightmass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse).The positive ion/linear mode was used, with an accelerating voltage of25 kV. Each spectrum was produced by accumulating data from ˜200 lasershots. External mass calibration was accomplished using purifiedproteins of known molecular masses.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

Definition of Terms

The terms used throughout this specification are defined as follows,unless otherwise limited in specific instances. As used in thespecification and the appended claims, the singular forms “a”, “an”, and“the” include plural referents unless the context clearly dictatesotherwise.

The term “half-life extending moiety” (i.e., F¹ or F² in Formula I)refers to a pharmaceutically acceptable moiety, domain, or “vehicle”covalently linked or conjugated to the toxin peptide, that prevents ormitigates in vivo proteolytic degradation or other activity-diminishingchemical modification of the toxin peptide, increases half-life or otherpharmacokinetic properties such as but not limited to increasing therate of absorption, reduces toxicity, improves solubility, increasesbiological activity and/or target selectivity of the toxin peptide withrespect to a target ion channel of interest, increasesmanufacturability, and/or reduces immunogenicity of the toxin peptide,compared to an unconjugated form of the toxin peptide.

By “PEGylated peptide” is meant a peptide or protein having apolyethylene glycol (PEG) moiety covalently bound to an amino acidresidue of the peptide itself or to a peptidyl or non-peptidyl linker(including but not limited to aromatic linkers) that is covalently boundto a residue of the peptide.

By “polyethylene glycol” or “PEG” is meant a polyalkylene glycolcompound or a derivative thereof, with or without coupling agents orderivatization with coupling or activating moieties (e.g., withaldehyde, hydroxysuccinimidyl, hydrazide, thiol, triflate, tresylate,azirdine, oxirane, orthopyridyl disulphide, vinylsulfone, iodoacetamideor a maleimide moiety). In accordance with the present invention, usefulPEG includes substantially linear, straight chain PEG, branched PEG, ordendritic PEG. (See, e.g., Merrill, U.S. Pat. No. 5,171,264; Harris etal., Multiarmed, monofunctional, polymer for coupling to molecules andsurfaces, U.S. Pat. No. 5,932,462; Shen, N-maleimidyl polymerderivatives, U.S. Pat. No. 6,602,498).

The term “peptibody” refers to molecules of Formula I in which F¹ and/orF² is an immunoglobulin Fc domain or a portion thereof, such as a CH2domain of an Fc, or in which the toxin peptide is inserted into a humanIgG1 Fc domain loop, such that F¹ and F² are each a portion of an Fcdomain with a toxin peptide inserted between them (See, e.g., FIGS.70-73 and Example 49 herein). Peptibodies of the present invention canalso be PEGylated as described further herein, at either an Fc domain orportion thereof, or at the toxin peptide(s) portion of the inventivecomposition, or both.

The term “native Fc” refers to molecule or sequence comprising thesequence of a non-antigen-binding fragment resulting from digestion ofwhole antibody, whether in monomeric or multimeric form. The originalimmunoglobulin source of the native Fc is preferably of human origin andcan be any of the immunoglobulins, although IgG1 or IgG2 are preferred.Native Fc's are made up of monomeric polypeptides that can be linkedinto dimeric or multimeric forms by covalent (i.e., disulfide bonds) andnon-covalent association. The number of intermolecular disulfide bondsbetween monomeric subunits of native Fc molecules ranges from 1 to 4depending on class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2,IgG3, IgA1, IgGA2). One example of a native Fc is a disulfide-bondeddimer resulting from papain digestion of an IgG (see Ellison et al.(1982), Nucleic Acids Res. 10: 4071-9). The term “native Fc” as usedherein is generic to the monomeric, dimeric, and multimeric forms.

The term “Fc variant” refers to a molecule or sequence that is modifiedfrom a native Fc but still comprises a binding site for the salvagereceptor, FcRn. Several published patent documents describe exemplary Fcvariants, as well as interaction with the salvage receptor. SeeInternational applications WO 97/34 631 (published 25 Sep. 1997; WO96/32 478, corresponding to U.S. Pat. No. 6,096,891, issued Aug. 1,2000, hereby incorporated by reference in its entirety; and WO 04/110472. Thus, the term “Fc variant” includes a molecule or sequence that ishumanized from a non-human native Fc. Furthermore, a native Fc comprisessites that can be removed because they provide structural features orbiological activity that are not required for the fusion molecules ofthe present invention. Thus, the term “Fc variant” includes a moleculeor sequence that lacks one or more native Fc sites or residues thataffect or are involved in (1) disulfide bond formation, (2)incompatibility with a selected host cell (3) N-terminal heterogeneityupon expression in a selected host cell, (4) glycosylation, (5)interaction with complement, (6) binding to an Fc receptor other than asalvage receptor, or (7) antibody-dependent cellular cytotoxicity(ADCC). Fc variants are described in further detail hereinafter.

The term “Fc domain” encompasses native Fc and Fc variant molecules andsequences as defined above. As with Fc variants and native Fc's, theterm “Fc domain” includes molecules in monomeric or multimeric form,whether digested from whole antibody or produced by other means.

The term “multimer” as applied to Fc domains or molecules comprising Fcdomains refers to molecules having two or more polypeptide chainsassociated covalently, noncovalently, or by both covalent andnon-covalent interactions. IgG molecules typically form dimers; IgM,pentamers; IgD, dimers; and IgA, monomers, dimers, trimers, ortetramers. One skilled in the art can form multimers by exploiting thesequence and resulting activity of the native Ig source of the Fc or byderivatizing (as defined below) such a native Fc.

The term “dimer” as applied to Fc domains or molecules comprising Fcdomains refers to molecules having two polypeptide chains associatedcovalently or non-covalently. Thus, exemplary dimers within the scope ofthis invention are as shown in FIG. 2.

The terms “derivatizing” and “derivative” or “derivatized” compriseprocesses and resulting compounds respectively in which (1) the compoundhas a cyclic portion; for example, cross-linking between cysteinylresidues within the compound; (2) the compound is cross-linked or has across-linking site; for example, the compound has a cysteinyl residueand thus forms cross-linked dimers in culture or in vivo; (3) one ormore peptidyl linkage is replaced by a non-peptidyl linkage; (4) theN-terminus is replaced by —NRR¹, NRC(O)R¹, —NRC(O)OR¹, —NRS(O)₂R¹,—NHC(O)NHR, a succinimide group, or substituted or unsubstitutedbenzyloxycarbonyl-NH—, wherein R and R¹ and the ring substituents are asdefined hereinafter; (5) the C-terminus is replaced by —C(O)R² or —NR³R⁴wherein R², R³ and R⁴ are as defined hereinafter; and (6) compounds inwhich individual amino acid moieties are modified through treatment withagents capable of reacting with selected side chains or terminalresidues. Derivatives are further described hereinafter.

The term “peptide” refers to molecules of 2 to about 80 amino acidresidues, with molecules of about 10 to about 60 amino acid residuespreferred and those of about 30 to about 50 amino acid residuess mostpreferred. Exemplary peptides can be randomly generated by any knownmethod, carried in a peptide library (e.g., a phage display library), orderived by digestion of proteins. In any peptide portion of theinventive compositions, for example a toxin peptide or a peptide linkermoiety described herein, additional amino acids can be included oneither or both of the N- or C-termini of the given sequence. Of course,these additional amino acid residues should not significantly interferewith the functional activity of the composition. “Toxin peptides”include peptides having the same amino acid sequence of a naturallyoccurring pharmacologically active peptide that can be isolated from avenom, and also include modified peptide analogs of such naturallyoccurring molecules. Examples of toxin peptides useful in practicing thepresent invention are listed in Tables 1-32. The toxin peptide (“P”, orequivalently shown as “P¹” in FIG. 2) comprises at least twointrapeptide disulfide bonds, as shown, for example, in FIG. 9.Accordingly, this invention concerns molecules comprising:

-   -   a) C¹-C³ and C²-C⁴ disulfide bonding in which C¹, C², C³, and C⁴        represent the order in which cysteine residues appear in the        primary sequence of the toxin peptide stated conventionally with        the N-terminus of the peptide on the left, with the first and        third cysteines in the amino acid sequence forming a disulfide        bond, and the second and fourth cysteines forming a disulfide        bond. Examples of toxin peptides with such a C¹-C³, C²-C⁴        disulfide bonding pattern include, but are not limited to,        apamin peptides, α-conopeptides, PnIA peptides, PnIB peptides,        and MII peptides, and analogs of any of the foregoing.    -   b) C¹-C⁶, C²-C⁴ and C³-C⁵ disulfide bonding in which, as        described above, C¹, C², C³, C⁴, C⁵ and C⁶ represent the order        of cysteine residues appearing in the primary sequence of the        toxin peptide stated conventionally with the N-terminus of the        peptide(s) on the left, with the first and sixth cysteines in        the amino acid sequence forming a disulfide bond, the second and        fourth cysteines forming a disulfide bond, and the third and        fifth cysteines forming a disulfide bond. Examples of toxin        peptides with such a C¹-C⁶, C²-C⁴, C³-C⁵disulfide bonding        pattern include, but are not limited to, ShK, BgK, HmK, AeKS,        AsK, and DTX1, and analogs of any of the foregoing.    -   c) C¹-C⁴, C²-C⁵ and C³-C⁶ disulfide bonding in which, as        described above, C¹, C², C³, C⁴, C⁵ and C⁶ represent the order        of cysteine residues appearing in the primary sequence of the        toxin peptide stated conventionally with the N-terminus of the        peptide(s) on the left, with the first and fourth cysteines in        the amino acid sequence forming a disulfide bond, the second and        fifth cysteines forming a disulfide bond, and the third and        sixth cysteines forming a disulfide bond. Examples of toxin        peptides with such a C¹-C⁴, C²-C⁵, C³-C⁶ disulfide bonding        pattern include, but are not limited to, ChTx, MgTx, OSK1, KTX1,        AgTx2, Pi2, Pi3, NTX, HgTx1, BeKM1, BmKTX, P01, BmKK6, Tc32,        Tc1, BmTx1, BmTX3, IbTx, P05, ScyTx, TsK, HaTx1, ProTX1, PaTX2,        Ptu1, ωGVIA, ωMVIIA, and SmIIIa, and analogs of any of the        foregoing.    -   d) C¹-C⁵, C²-C⁶, C³-C⁷, and C⁴-C⁸ disulfide bonding in which C¹,        C², C³, C⁴, C⁵, C⁶, C⁷ and C⁸ represent the order of cysteine        residues appearing in the primary sequence of the toxin peptide        stated conventionally with the N-terminus of the peptide(s) on        the left, with the first and fifth cysteines in the amino acid        sequence forming a disulfide bond, the second and sixth        cysteines forming a disulfide bond, the third and seventh        cysteines forming a disulfide bond, and the fourth and eighth        cysteines forming a disulfide bond. Examples of toxin peptides        with such a C¹-C⁵, C²-C⁶, C³-C⁷, C⁴-C⁸ disulfide bonding pattern        include, but are not limited to, Anuoroctoxin (AnTx), Pi1,        HsTx1, MTX (P12A, P20A), and Pi4 peptides, and analogs of any of        the foregoing.    -   e) C¹-C⁴, C²-C⁶, C³-C⁷, and C⁵-C⁸ disulfide bonding in which C¹,        C², C³, C⁴, C⁵, C⁶, C⁷ and C⁸ represent the order of cysteine        residues appearing in the primary sequence of the toxin peptide        stated conventionally with the N-terminus of the peptide(s) on        the left, with the first and fourth cysteines in the amino acid        sequence forming a disulfide bond, the second and sixth        cysteines forming a disulfide bond, the third and seventh        cysteines forming a disulfide bond, and the fifth and eighth        cysteines forming a disulfide bond. Examples of toxin peptides        with such a C¹-C⁴, C²-C⁶, C³-C⁷, C⁵-C⁸ disulfide bonding pattern        include, but are not limited to, Chlorotoxin, Bm-12b, and, and        analogs of either.    -   f) C¹-C⁵, C²-C⁶, C³-C⁴, and C⁷-C⁸ disulfide bonding in which C¹,        C², C³, C⁴, C⁵, C⁶, C⁷ and C⁸ represent the order of cysteine        residues appearing in the primary sequence of the toxin peptide        stated conventionally with the N-terminus of the peptide(s) on        the left, with the first and fifth cysteines in the amino acid        sequence forming a disulfide bond, the second and sixth        cysteines forming a disulfide bond, the third and fourth        cysteines forming a disulfide bond, and the seventh and eighth        cysteines forming a disulfide bond. Examples of toxin peptides        with such a C¹-C⁵, C²-C⁶, C³-C⁴, C⁷-C⁸ disulfide bonding pattern        include, but are not limited to, Maurotoxin peptides and analogs        thereof.

The term “randomized” as used to refer to peptide sequences refers tofully random sequences (e.g., selected by phage display methods) andsequences in which one or more residues of a naturally occurringmolecule is replaced by an amino acid residue not appearing in thatposition in the naturally occurring molecule. Exemplary methods foridentifying peptide sequences include phage display, E. coli display,ribosome display, yeast-based screening, RNA-peptide screening, chemicalscreening, rational design, protein structural analysis, and the like.

The term “pharmacologically active” means that a substance so describedis determined to have activity that affects a medical parameter (e.g.,blood pressure, blood cell count, cholesterol level) or disease state(e.g., cancer, autoimmune disorders). Thus, pharmacologically activepeptides comprise agonistic or mimetic and antagonistic peptides asdefined below.

The terms “-mimetic peptide” and “-agonist peptide” refer to a peptidehaving biological activity comparable to a naturally occurring toxinpeptide molecule, e.g., naturally occurring ShK toxin peptide. Theseterms further include peptides that indirectly mimic the activity of anaturally occurring toxin peptide molecule, such as by potentiating theeffects of the naturally occurring molecule.

The term “-antagonist peptide” or “inhibitor peptide” refers to apeptide that blocks or in some way interferes with the biologicalactivity of a receptor of interest, or has biological activitycomparable to a known antagonist or inhibitor of a receptor of interest(such as, but not limited to, an ion channel).

The term “acidic residue” refers to amino acid residues in D- or L-formhaving sidechains comprising acidic groups. Exemplary acidic residuesinclude D and E.

The term “amide residue” refers to amino acids in D- or L-form havingsidechains comprising amide derivatives of acidic groups. Exemplaryresidues include N and Q.

The term “aromatic residue” refers to amino acid residues in D- orL-form having sidechains comprising aromatic groups. Exemplary aromaticresidues include F, Y, and W.

The term “basic residue” refers to amino acid residues in D- or L-formhaving sidechains comprising basic groups. Exemplary basic residuesinclude H, K, R, N-methyl-arginine, ω-aminoarginine, ω-methyl-arginine,1-methyl-histidine, 3-methyl-histidine, and homoarginine (hR) residues.

The term “hydrophilic residue” refers to amino acid residues in D- orL-form having sidechains comprising polar groups. Exemplary hydrophilicresidues include C, S, T, N, Q, D, E, K, and citrulline (Cit) residues.

The term “nonfunctional residue” refers to amino acid residues in D- orL-form having sidechains that lack acidic, basic, or aromatic groups.Exemplary nonfunctional amino acid residues include M, G, A, V, I, L andnorleucine (Nle).

The term “neutral polar residue” refers to amino acid residues in D- orL-form having sidechains that lack basic, acidic, or polar groups.Exemplary neutral polar amino acid residues include A, V, L, I, P, W, M,and F.

The term “polar hydrophobic residue” refers to amino acid residues in D-or L-form having sidechains comprising polar groups. Exemplary polarhydrophobic amino acid residues include T, G, S, Y, C, Q, and N.

The term “hydrophobic residue” refers to amino acid residues in D- orL-form having sidechains that lack basic or acidic groups. Exemplaryhydrophobic amino acid residues include A, V, L, I, P, W, M, F, T, G, S,Y, C, Q, and N.

In some useful embodiments of the compositions of the invention, theamino acid sequence of the toxin peptide is modified in one or more waysrelative to a native toxin peptide sequence of interest, such as, butnot limited to, a native ShK or OSK1 sequence, their peptide analogs, orany other toxin peptides having amino acid sequences as set for in anyof Tables 1-32. The one or more useful modifications can include aminoacid additions or insertions, amino acid deletions, peptide truncations,amino acid substitutions, and/or chemical derivatization of amino acidresidues, accomplished by known chemical techniques. Such modificationscan be, for example, for the purpose of enhanced potency, selectivity,and/or proteolytic stability, or the like. Those skilled in the art areaware of techniques for designing peptide analogs with such enhancedproperties, such as alanine scanning, rational design based on alignmentmediated mutagenesis using known toxin peptide sequences and/ormolecular modeling. For example, ShK analogs can be designed to removeprotease cleavage sites (e.g., trypsin cleavage sites at K or R residuesand/or chymotrypsin cleavage sites at F, Y, or W residues) in a ShKpeptide- or ShK analog-containing composition of the invention, basedpartially on alignment mediated mutagenesis using HmK (see, e.g., FIG.6) and molecular modeling. (See, e.g., Kalman et al., ShK-Dap22, apotent Kv1.3-specific immunosuppressive polypeptide, J. Biol. Chem.273(49):32697-707 (1998); Kem et al., U.S. Pat. No. 6,077,680; Mouhat etal., OsK1 derivatives, WO 2006/002850 A2)).

The term “protease” is synonymous with “peptidase”. Proteases comprisetwo groups of enzymes: the endopeptidases which cleave peptide bonds atpoints within the protein, and the exopeptidases, which remove one ormore amino acids from either N- or C-terminus respectively. The term“proteinase” is also used as a synonym for endopeptidase. The fourmechanistic classes of proteinases are: serine proteinases, cysteineproteinases, aspartic proteinases, and metallo-proteinases. In additionto these four mechanistic classes, there is a section of the enzymenomenclature which is allocated for proteases of unidentified catalyticmechanism. This indicates that the catalytic mechanism has not beenidentified.

Cleavage subsite nomenclature is commonly adopted from a scheme createdby Schechter and Berger (Schechter I. & Berger A., On the size of theactive site in proteases. I. Papain, Biochemical and BiophysicalResearch Communication, 27:157 (1967); Schechter I. & Berger A., On theactive site of proteases. 3. Mapping the active site of papain; specificinhibitor peptides of papain, Biochemical and Biophysical ResearchCommunication, 32:898 (1968)). According to this model, amino acidresidues in a substrate undergoing cleavage are designated P1, P2, P3,P4 etc. in the N-terminal direction from the cleaved bond. Likewise, theresidues in the C-terminal direction are designated P1′, P2′, P3′, P4′.etc.

The skilled artisan is aware of a variety of tools for identifyingprotease binding or protease cleavage sites of interest. For example,the PeptideCutter software tool is available through the ExPASy (ExpertProtein Analysis System) proteomics server of the Swiss Institute ofBioinformatics (SIB; www.expasy.org/tools/peptidecutter). PeptideCuttersearches a protein sequence from the SWISS-PROT and/or TrEMBL databasesor a user-entered protein sequence for protease cleavage sites. Singleproteases and chemicals, a selection or the whole list of proteases andchemicals can be used. Different forms of output of the results areavailable: tables of cleavage sites either grouped alphabeticallyaccording to enzyme names or sequentially according to the amino acidnumber. A third option for output is a map of cleavage sites. Thesequence and the cleavage sites mapped onto it are grouped in blocks,the size of which can be chosen by the user. Other tools are also knownfor determining protease cleavage sites. (E.g., Turk, B. et al.,Determination of protease cleavage site motifs using mixture-basedoriented peptide libraries, Nature Biotechnology, 19:661-667 (2001);Barrett A. et al., Handbook of proteolytic enzymes, Academic Press(1998)).

The serine proteinases include the chymotrypsin family, which includesmammalian protease enzymes such as chymotrypsin, trypsin or elastase orkallikrein. The serine proteinases exhibit different substratespecificities, which are related to amino acid substitutions in thevarious enzyme subsites interacting with the substrate residues. Someenzymes have an extended interaction site with the substrate whereasothers have a specificity restricted to the P1 substrate residue.

Trypsin preferentially cleaves at R or K in position P1. A statisticalstudy carried out by Keil (1992) described the negative influences ofresidues surrounding the Arg- and Lys-bonds (i.e. the positions P2 andP1′, respectively) during trypsin cleavage. (Keil, B., Specificity ofproteolysis, Springer-Verlag Berlin-Heidelberg-NewYork, 335 (1992)). Aproline residue in position P1′ normally exerts a strong negativeinfluence on trypsin cleavage. Similarly, the positioning of R and K inP1′ results in an inhibition, as well as negatively charged residues inpositions P2 and P1′.

Chymotrypsin preferentially cleaves at a W, Y or F in position P1 (highspecificity) and to a lesser extent at L, M or H residue in position P1.(Keil, 1992). Exceptions to these rules are the following: When W isfound in position P1, the cleavage is blocked when M or P are found inposition P1′ at the same time. Furthermore, a proline residue inposition P1′ nearly fully blocks the cleavage independent of the aminoacids found in position P1. When an M residue is found in position P1,the cleavage is blocked by the presence of a Y residue in position P1′.Finally, when H is located in position P1, the presence of a D, M or Wresidue also blocks the cleavage.

Membrane metallo-endopeptidase (NEP; neutral endopeptidase,kidney-brush-border neutral proteinase, enkephalinase, EC 3.4.24.11)cleaves peptides at the amino side of hydrophobic amino acid residues.(Connelly, J C et al., Neutral Endopeptidase 24.11 in Human Neutrophils:Cleavage of Chemotactic Peptide, PNAS, 82(24):8737-8741 (1985)).

Thrombin preferentially cleaves at an R residue in position P1. (Keil,1992). The natural substrate of thrombin is fibrinogen. Optimum cleavagesites are when an R residue is in position P1 and Gly is in position P2and position P1′. Likewise, when hydrophobic amino acid residues arefound in position P4 and position P3, a proline residue in position P2,an R residue in position P1, and non-acidic amino acid residues inposition P1′ and position P2′. A very important residue for its naturalsubstrate fibrinogen is a D residue in P10.

Caspases are a family of cysteine proteases bearing an active site witha conserved amino acid sequence and which cleave peptides specificallyfollowing D residues. (Earnshaw W C et al., Mammalian caspases:Structure, activation, substrates, and functions during apoptosis,Annual Review of Biochemistry, 68:383-424 (1999)).

The Arg-C proteinase preferentially cleaves at an R residue in positionP1. The cleavage behavior seems to be only moderately affected byresidues in position P1′. (Keil, 1992). The Asp-N endopeptidase cleavesspecifically bonds with a D residue in position P1′. (Keil, 1992).

The foregoing is merely exemplary and by no means an exhaustivetreatment of knowledge available to the skilled artisan concerningprotease binding and/or cleavage sites that the skilled artisan may beinterested in eliminating in practicing the invention.

In other examples, a toxin peptide amino acid sequence modified from anaturally occurring toxin peptide amino acid sequence includes at leastone amino acid residue inserted or substituted therein, relative to theamino acid sequence of the native toxin peptide sequence of interest, inwhich the inserted or substituted amino acid residue has a side chaincomprising a nucleophilic or electrophilic reactive functional group bywhich the peptide is conjugated to a linker or half-life extendingmoiety. In accordance with the invention, useful examples of such anucleophilic or electrophilic reactive functional group include, but arenot limited to, a thiol, a primary amine, a seleno, a hydrazide, analdehyde, a carboxylic acid, a ketone, an aminooxy, a masked (protected)aldehyde, or a masked (protected) keto functional group. Examples ofamino acid residues having a side chain comprising a nucleophilicreactive functional group include, but are not limited to, a lysineresidue, an α,β-diaminopropionic acid residue, an α,γ-diaminobutyricacid residue, an ornithine residue, a cysteine, a homocysteine, aglutamic acid residue, an aspartic acid residue, or a selenocysteineresidue.

In further describing toxin peptides herein, a one-letter abbreviationsystem is frequently applied to designate the identities of the twenty“canonical” amino acid residues generally incorporated into naturallyoccurring peptides and proteins (Table 1A). Such one-letterabbreviations are entirely interchangeable in meaning with three-letterabbreviations, or non-abbreviated amino acid names. Within theone-letter abbreviation system used herein, an uppercase letterindicates a L-amino acid, and a lower case letter indicates a D-aminoacid, unless otherwise noted herein. For example, the abbreviation “R”designates L-arginine and the abbreviation “r” designates D-arginine.

TABLE 1A One-letter abbreviations for the canonical amino acids.Three-letter abbreviations are in parentheses. Alanine (Ala) A Glutamine(Gln) Q Leucine (Leu) L Serine (Ser) S Arginine (Arg) R Glutamic Acid(Glu) E Lysine (Lys) K Threonine (Thr) T Asparagine (Asn) N Glycine(Gly) G Methionine (Met) M Tryptophan (Trp) W Aspartic Acid (Asp) DHistidine (His) H Phenylalanine (Phe) F Tyrosine (Tyr) Y Cysteine (Cys)C Isoleucine (Ile) I Proline (Pro) P Valine (Val) V

An amino acid substitution in an amino acid sequence is typicallydesignated herein with a one-letter abbreviation for the amino acidresidue in a particular position, followed by the numerical amino acidposition relative to the native toxin peptide sequence of interest,which is then followed by the one-letter symbol for the amino acidresidue substituted in. For example, “T30D” symbolizes a substitution ofa threonine residue by an aspartate residue at amino acid position 30,relative to a hypothetical native toxin peptide sequence. By way offurther example, “R18hR” or “R18Cit” indicates a substitution of anarginine residue by a homoarginine or a citrulline residue,respectively, at amino acid position 18, relative to the hypotheticalnative toxin peptide. An amino acid position within the amino acidsequence of any particular toxin peptide (or peptide analog) describedherein may differ from its position relative to the native sequence,i.e., as determined in an alignment of the N-terminal or C-terminal endof the peptide's amino acid sequence with the N-terminal or C-terminalend, as appropriate, of the native toxin peptide sequence. For example,amino acid position 1 of the sequenceSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC(ShK(2-35); SEQ ID NO:92), aN-terminal truncation of the native ShK sequence, thus aligned with theC-terminal of native ShK(1-35) (SEQ ID NO:5), corresponds to amino acidposition 2 relative to the native sequence, and amino acid position 34of SEQ ID NO:92 corresponds to amino acid position 35 relative to thenative sequence (SEQ ID NO:5).

In certain embodiments of the present invention, amino acidsubstitutions encompass, non-canonical amino acid residues, which caninclude naturally rare (in peptides or proteins) amino acid residues orunnatural amino acid residues. Non-canonical amino acid residues can beincorporated into the peptide by chemical peptide synthesis rather thanby synthesis in biological systems, such as recombinantly expressingcells, or alternatively the skilled artisan can employ known techniquesof protein engineering that use recombinantly expressing cells. (See,e.g., Link et al., Non-canonical amino acids in protein engineering,Current Opinion in Biotechnology, 14(6):603-609 (2003)). The term“non-canonical amino acid residue” refers to amino acid residues in D-or L-form that are not among the 20 canonical amino acids generallyincorporated into naturally occurring proteins, for example, β-aminoacids, homoamino acids, cyclic amino acids and amino acids withderivatized side chains. Examples include (in the L-form or D-form):citrulline (Cit), homocitrulline (hCit), N-methylcitrulline (NMeCit),N-methylhomocitrulline (NMeHoCit), ornithine (Orn or O),N-Methylornithine (NMeOrn), sarcosine (Sar), homolysine (hK or Hlys),homoarginine (hR or hArg), homoglutamine (hQ), N-methylarginine (NMeR),N-methylleucine (NMeL), N-methylhomolysine (NMeHoK), N-methylglutamine(NMeQ), norleucine (Nle), norvaline (Nva),1,2,3,4-tetrahydroisoquinoline (Tic), nitrophenylalanine (nitrophe),aminophenylalanine (aminophe), benzylphenyalanine (benzylphe),γ-carboxyglutamic acid (γ-carboxyglu), hydroxyproline (hydroxypro),p-carboxyl-phenylalanine (Cpa), α-aminoadipic acid (Aad), acetylarginine(acetylarg), α,β-diaminopropionoic acid (Dpr), α,γ-diaminobutyric acid(Dab), diaminopropionic acid (Dap), β-(1-Naphthyl)-alanine (1Na1),β-(2-Naphthyl)-alanine (2Na1), cyclohexylalanine (Cha),4-methyl-phenylalanine (MePhe), β,β-diphenyl-alanine (BiPhA),aminobutyric acid (Abu), 4-phenyl-phenylalanine (4Bip),α-amino-isobutyric acid (Aib), and derivatized forms of any of these asdescribed herein. Nomenclature and Symbolism for Amino Acids andPeptides by the UPAC-IUB Joint Commission on Biochemical Nomenclature(JCBN) have been published in the following documents: Biochem. J.,1984, 219, 345-373; Eur. J. Biochem., 1984, 138, 9-37; 1985, 152, 1;1993, 213, 2; Internat. J. Pept. Prot. Res., 1984, 24, following p 84;J. Biol. Chem., 1985, 260, 14-42; Pure Appl. Chem., 1984, 56, 595-624;Amino Acids and Peptides, 1985, 16, 387-410; Biochemical Nomenclatureand Related Documents, 2nd edition, Portland Press, 1992, pages 39-69].

As stated herein, in accordance with the present invention, peptideportions of the inventive compositions, such as the toxin peptide or apeptide linker, can also be chemically derivatized at one or more aminoacid residues. Peptides that contain derivatized amino acid residues canbe synthesized by known organic chemistry techniques. “Chemicalderivative” or “chemically derivatized” in the context of a peptiderefers to a subject peptide having one or more residues chemicallyderivatized by reaction of a functional side group. Such derivatizedmolecules include, for example, those molecules in which free aminogroups have been derivatized to form amine hydrochlorides, p-toluenesulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups,chloroacetyl groups or formyl groups. Free carboxyl groups may bederivatized to form salts, methyl and ethyl esters or other types ofesters or hydrazides. Free hydroxyl groups may be derivatized to formO-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine maybe derivatized to form N-im-benzylhistidine. Also included as chemicalderivatives are those peptides which contain one or more naturallyoccurring amino acid derivatives of the twenty canonical amino acids,whether in L- or D-form. For example, 4-hydroxyproline may besubstituted for proline; 5-hydroxylysine maybe substituted for lysine;3-methylhistidine may be substituted for histidine; homoserine may besubstituted for serine; and ornithine may be substituted for lysine.

In some embodiments of the present invention, basic residues (e.g.,lysine) of the toxin peptide of interest can be replaced with otherresidues (nonfunctional residues preferred). Such molecules will be lessbasic than the molecules from which they are derived and otherwiseretain the activity of the molecules from which they are derived, whichcan result in advantages in stability and immunogenicity; the presentinvention should not, however, be limited by this theory.

Additionally, physiologically acceptable salts of the inventivecompositions are also encompassed, including when the inventivecompositons are referred to herein as “molecules” or “compounds.”. By“physiologically acceptable salts” is meant any salts that are known orlater discovered to be pharmaceutically acceptable. Some examples are:acetate; trifluoroacetate; hydrohalides, such as hydrochloride andhydrobromide; sulfate; citrate; maleate; tartrate; glycolate; gluconate;succinate; mesylate; besylate; and oxalate salts.

Structure of Compounds:

In General. Recombinant proteins have been developed as therapeuticagents through, among other means, covalent attachment to half-lifeextending moieties. Such moieties include the “Fc” domain of anantibody, as is used in Enbrel® (etanercept), as well as biologicallysuitable polymers (e.g., polyethylene glycol, or “PEG”), as is used inNeulasta® (pegfilgrastim). Feige et al. described the use of suchhalf-life extenders with peptides in U.S. Pat. No. 6,660,843, issuedDec. 9, 2003 (hereby incorporated by reference in its entirety).

The present inventors have determined that molecules of thisinvention—peptides of about 80 amino acids or less with at least twointrapeptide disulfide bonds—possess therapeutic advantages whencovalently attached to half-life extending moieties. Molecules of thepresent invention can further comprise an additional pharmacologicallyactive, covalently bound peptide, which can be bound to the half-lifeextending moiety (F¹ and/or F²) or to the peptide portion (P).Embodiments of the inventive compositions containing more than onehalf-life extending moiety (F¹ and F²) include those in which F¹ and F²are the same or different half-life extending moieties. Examples (withor without a linker between each domain) include structures asillustrated in FIG. 75 as well as the following embodiments (and othersdescribed herein and in the working Examples):

20KPEG—toxin peptide—Fc domain, consistent with the formula[(F¹)₁—(X²)₁—(F²)₁];

20KPEG—toxin peptide—Fc CH2 domain, consistent with the formula[(F¹)₁—(X²)₁—(F²)₁];

20KPEG—toxin peptide—HSA, consistent with the formula[(F¹)₁—(X²)₁—(F²)₁];

20KPEG—Fc domain—toxin peptide, consistent with the formula[(F¹)₁—(F²)₁—(X³)₁];

20KPEG—Fc CH2 domain—toxin peptide, consistent with the formula[(F¹)₁—(F²)₁—(X³)₁]; and

20KPEG—HSA—toxin peptide, consistent with the formula[(F¹)₁—(F²)₁—(X³)₁].

Toxin Peptides. Any number of toxin peptides (i.e., “P”, or equivalentlyshown as “P¹” in FIG. 2) can be used in conjunction with the presentinvention. Of particular interest are the toxin peptides ShK, HmK, MgTx,AgTx2, OsK1 (also referred to as “OSK1”), Agatoxins, and HsTx1, as wellas modified analogs of these, and other peptides that mimic the activityof such toxin peptides. As stated herein above, if more than one toxinpeptide “P” is present in the inventive composition, “P” can beindependently the same or different from any other toxin peptide(s) alsopresent in the inventive composition. For example, in a compositionhaving the formula P-(L)_(g)-F¹-(L)_(f)-P, both of the toxin peptides,“P”, can be the same peptide analog of ShK, different peptide analogs ofShK, or one can be a peptide analog of ShK and the other a peptideanalog of OSK1.

In some embodiments of the invention, other peptides of interest areespecially useful in molecules having additional features over themolecules of structural Formula I. In such molecules, the molecule ofFormula I further comprises an additional pharmacologically active,covalently bound peptide, which is an agonistic peptide, an antagonisticpeptide, or a targeting peptide; this peptide can be conjugated to F¹ orF² or P. Such agonistic peptides have activity agonistic to the toxinpeptide but are not required to exert such activity by the samemechanism as the toxin peptide. Peptide antagonists are also useful inembodiments of the invention, with a preference for those with activitythat can be complementary to the activity of the toxin peptide.Targeting peptides are also of interest, such as peptides that directthe molecule to particular cell types, organs, and the like. Theseclasses of peptides can be discovered by methods described in thereferences cited in this specification and other references. Phagedisplay, in particular, is useful in generating toxin peptides for usein the present invention. Affinity selection from libraries of randompeptides can be used to identify peptide ligands for any site of anygene product. Dedman et al. (1993), J. Biol. Chem. 268: 23025-30. Phagedisplay is particularly well suited for identifying peptides that bindto such proteins of interest as cell surface receptors or any proteinshaving linear epitopes. Wilson et al. (1998), Can. J. Microbiol. 44:313-29; Kay et al. (1998), Drug Disc. Today 3: 370-8. Such proteins areextensively reviewed in Herz et al. (1997), J. Receptor and SignalTransduction Res. 17(5): 671-776, which is hereby incorporated byreference in its entirety. Such proteins of interest are preferred foruse in this invention.

Particularly preferred peptides appear in the following tables. Thesepeptides can be prepared by methods disclosed in the art or as describedhereinafter. Single letter amino acid abbreviations are used. Unlessotherwise specified, each X is independently a nonfunctional residue.

TABLE 1 Kv1.3 inhibitor peptide sequences Short- hand SEQ designa- IDSequence/structure tion NO: LVKCRGTSDCGRPCQQQTGCPNSKCINRMCKCYGC Pi1 21TISCTNPKQCYPHCKKETGYPNAKCMNRKCKCFGR Pi2 17TISCTNEKQCYPHCKKETGYPNAKCMNRKCKCFGR Pi3 18IEAIRCGGSRDCYRPCQKRTGCPNAKCINKTCKCYGCS Pi4 19ASCRTPKDCADPCRKETGCPYGKCMNRKCKCNRC HsTx1 61GVPINVSCTGSPQCIKPCKDAGMRFGKCMNRKCHCTPK AgTx2 23GVPINVKCTGSPQCLKPCKDAGMRFGKCINGKCHCTPK AgTx1 85GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK OSK1 25ZKECTGPQHCTNFCRKNKCTHGKCMNRKCKCFNCK Anuroc- 62 toxinTIINVKCTSPKQCSKPCKELYGSSAGAKCMNGKCKCYNN NTX 30TVIDVKCTSPKQCLPPCKAQFGIRAGAKGMNGKCKCYPH HgTx1 27QFTNVSCTTSKECWSVCQRLHNTSRGKCMNKKCRGYS ChTx 36VFINAKCRGSPECLPKCKEAIGKAAGKCMNGKCKCYP Titys- 86 toxin-KaVCRDWFKETACRHAKSLGNCRTSQKYRANCAKTCELC BgK 9VGINVKCKHSGQCLKPCKDAGMRFGKCINGKCDCTPKG BmKTX 26QFTDVKCTGSKQCWPVCKQMFGKPNGKCMNGKCRCYS BmTx1 40VFINVKCRGSKECLPACKAAVGKAAGKCMNGKCKCYP Tc30 87TGPQTTCQAAMCEAGCKGLGKSMESCQGDTCKCKA Tc32 13

TABLE 2 ShK peptide and ShK peptide analog sequences Short- hand SEQdesig- ID Sequence/structure nation NO:RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK 5RSCIDTIPKSRCTAFQSKHSMKYRLSFCRKTSGTC ShK- 88 S17/ S32RSSIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTS ShK- 89 S3/S35SSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-S1 90 (N-acetylarg) ShK-N- 91SCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ace- tyl- arg1 SCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-d1 92  CIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-d2 93ASCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-A1 94RSCADTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-A4 95RSCADTIPKSRCTAAQCKHSMKYRLSFCRKTCGTC ShK- 96 A4/A15RSCADTIPKSRCTAAQCKHSMKYRASFCRKTCGTC ShK- 97 A4/ A15/ A25RSCIDAIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-A6 98RSCIDTAPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-A7 99RSCIDTIAKSRCTAFQCKHSMKYRLSFCRKTCGTC Shk-A8 100RSCIDTIPASRCTAFQCKHSMKYRLSFCRKTCGTC ShK-A9 101RSCIDTIPESRCTAFQCKHSMKYRLSFCRKTCGTC ShK-E9 102RSCIDTIPQSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-Q9 103RSCIDTIPKARCTAFQCKHSMKYRLSFCRKTCGTC ShK- 104 A10RSCIDTIPKSACTAFQCKHSMKYRLSFCRKTCGTC ShK- 105 A11RSCIDTIPKSECTAFQCKHSMKYRLSFCRKTCGTC ShK- 106 E11RSCIDTIPKSQCTAFQCKHSMKYRLSFCRKTCGTC ShK- 107 Q11RSCIDTIPKSRCAAFQCKHSMKYRLSFCRKTCGTC ShK- 108 A13RSCIDTIPKSRCTAAQCKHSMKYRLSFCRKTCGTC ShK- 109 A15RSCIDTIPKSRCTAWQCKHSMKYRLSFCRKTCGTC ShK- 110 W15RSCIDTIPKSRCTAX^(s15)QCKHSMKYRLSFCRKTCGTC ShK- 111 X15RSCIDTIPKSRCTAAQCKHSMKYRASFCRKTCGTC ShK- 112 A15/ A25RSCIDTIPKSRCTAFACKHSMKYRLSFCRKTCGTC ShK- 113 A16RSCIDTIPKSRCTAFECKHSMKYRLSFCRKTCGTC ShK- 114 E16RSCIDTIPKSRCTAFQCAHSMKYRLSFCRKTCGTC ShK- 115 A18RSCIDTIPKSRCTAFQCEHSMKYRLSFCRKTCGTC ShK- 116 E18RSCIDTIPKSRCTAFQCKASMKYRLSFCRKTCGTC ShK- 117 A19RSCIDTIPKSRCTAFQCKKSMKYRLSFCRKTCGTC ShK- 118 K19RSCIDTIPKSRCTAFQCKHAMKYRLSFCRKTCGTC ShK- 119 A20RSCIDTIPKSRCTAFQCKHSAKYRLSFCRKTCGTC ShK- 120 A21RSCIDTIPKSRCTAFQCKHSX^(s21)KYRLSFCRKTCGTC ShK- 121 X21RSCIDTIPKSRCTAFQCKHS(norleu) ShK- 122 KYRLSFCRKTCGTC Nle21RSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC ShK- 123 A22RSCIDTIPKSRCTAFQCKHSMEYRLSFCRKTCGTC ShK- 124 E22RSCIDTIPKSRCTAFQCKHSMRYRLSFCRKTCGTC ShK- 125 R22RSCIDTIPKSRCTAFQCKHSMX^(s22)YRLSFCRKTCGTC ShK- 126 X22RSCIDTIPKSRCTAFQCKHSM(norleu) ShK- 127 YRLSFCRKTCGTC Nle22RSCIDTIPKSRCTAFQCKHSM(orn) ShK- 128 YRLSFCRKTCGTC Orn22RSCIDTIPKSRCTAFQCKHSM(homocit) ShK- 129 YRLSFCRKTCGTC Homo- cit22RSCIDTIPKSRCTAFQCKHSM(diamino- ShK- 130 propionic)YRLSFCRKTCGTC Di-amino- propi- onic22 RSCIDTIPKSRCTAFQCKHSMKARLSFCRKTCGTC ShK- 131 A23RSCIDTIPKSRCTAFQCKHSMKSRLSFCRKTCGTC ShK- 132 S23RSCIDTIPKSRCTAFQCKHSMKFRLSFCRKTCGTC ShK- 133 F23RSCIDTIPKSRCTAFQCKHSMKX^(s23)RLSFCRKTCGTC ShK- 134 X23RSCIDTIPKSRCTAFQCKHSMK(nitrophe) ShK- 135 RLSFCRKTCGTC Nitro- phe23RSCIDTIPKSRCTAFQCKHSMK(aminophe) ShK- 136 RLSFCRKTCGTC Amino- phe23RSCIDTIPKSRCTAFQCKHSMK(benzylphe) ShK- 137 RLSFCRKTCGTC Ben- zyl- phe23RSCIDTIPKSRCTAFQCKHSMKYALSFCRKTCGTC ShK- 138 A24RSCIDTIPKSRCTAFQCKHSMKYELSFCRKTCGTC ShK- 139 E24RSCIDTIPKSRCTAFQCKHSMKYRASFCRKTCGTC ShK- 140 A25RSCIDTIPKSRCTAFQCKHSMKYRLAFCRKTCGTC ShK- 141 A26RSCIDTIPKSRCTAFQCKHSMKYRLSACRKTCGTC ShK- 142 A27RSCIDTIPKSRCTAFQCKHSMKYRLSX^(s27)CRKTCGTC ShK- 143 X27RSCIDTIPKSRCTAFQCKHSMKYRLSFCAKTCGTC ShK- 144 A29RSCIDTIPKSRCTAFQCKHSMKYRLSFCRATCGTC ShK- 145 A30RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKACGTC ShK- 146 A31RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGAC ShK- 147 A34 SCADTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK- 148 A4d1 SCADTIPKSRCTAAQCKHSMKYRLSFCRKTCGTC ShK- 149 A4/ A15d1 SCADTIPKSRCTAAQCKHSMKYRASFCRKTCGTC ShK- 150 A4/ A15/ A25 d1 SCIDAIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-A6 151 d1 SCIDTAPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-A7 152 d1 SCIDTIAKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-A8 153 d1 SCIDTIPASRCTAFQCKHSMKYRLSFCRKTCGTC ShK-A9 154 d1 SCIDTIPESRCTAFQCKHSMKYRLSFCRKTCGTC ShK-E9 155 d1 SCIDTIPQSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-Q9 156 d1 SCIDTIPKARCTAFQCKHSMKYRLSFCRKTCGTC ShK- 157 A10 d1 SCIDTIPKSACTAFQCKHSMKYRLSFCRKTCGTC ShK- 158 A11 d1 SCIDTIPKSECTAFQCKHSMKYRLSFCRKTCGTC ShK- 159 E11 d1 SCIDTIPKSQCTAFQCKHSMKYRLSFCRKTCGTC ShK- 160 Q11 d1 SCIDTIPKSRCAAFQCKHSMKYRLSFCRKTCGTC ShK- 161 A13 d1 SCIDTIPKSRCTAAQCKHSMKYRLSFCRKTCGTC ShK- 162 A15 d1 SCIDTIPKSRCTAWQCKHSMKYRLSFCRKTCGTC ShK- 163 W15 d1 SCIDTIPKSRCTAX^(s15)QCKHSMKYRLSFCRKTCGTC ShK- 164 X15 d1 SCIDTIPKSRCTAAQCKHSMKYRASFCRKTCGTC ShK- 165 A15/ A25 d1 SCIDTIPKSRCTAFACKHSMKYRLSFCRKTCGTC ShK- 166 A16 d1 SCIDTIPKSRCTAFECKHSMKYRLSFCRKTCGTC ShK- 167 E16 d1 SCIDTIPKSRCTAFQCAHSMKYRLSFCRKTCGTC ShK- 168 A18 d1 SCIDTIPKSRCTAFQCEHSMKYRLSFCRKTCGTC ShK- 169 E18 d1 SCIDTIPKSRCTAFQCKASMKYRLSFCRKTCGTC ShK- 170 A19 d1 SCIDTIPKSRCTAFQCKKSMKYRLSFCRKTCGTC ShK- 171 K19 d1 SCIDTIPKSRCTAFQCKHAMKYRLSFCRKTCGTC ShK- 172 A20 d1 SCIDTIPKSRCTAFQCKHSAKYRLSFCRKTCGTC ShK- 173 A21 d1 SCIDTIPKSRCTAFQCKHSX^(s21)KYRLSFCRKTCGTC ShK- 174 X21 d1SCIDTIPKSRCTAFQCKHS(norleu) ShK- 175 KYRLSPCRKTCGTC Nle21 d1 SCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC ShK- 176 A22 d1 SCIDTIPKSRCTAFQCKHSMEYRLSFCRKTCGTC ShK- 177 E22 d1 SCIDTIPKSRCTAFQCKHSMRYRLSFCRKTCGTC ShK- 178 R22 d1 SCIDTIPKSRCTAFQCKHSMX^(s22)YRLSFCRKTCGTC ShK- 179 X22 d1SCIDTIPKSRCTAFQCKHSM(norleu) ShK- 180 YRLSFCRKTCGTC Nle22 d1SCIDTIPKSRCTAFQCKHSM(orn) ShK- 181 YRLSFCRKTCGTC Orn22 d1SCIDTIPKSRCTAFQCKHSM(homocit) ShK- 182 YRLSFCRKTCGTC Homo- cit22 d1SCIDTIPKSRCTAFQCKHSM(diamino- ShK- 183 propionic) YRLSF Di- CRKTCGTCamino- propi- onic22 d1  SCIDTIPKSRCTAFQCKHSMKARLSFCRKTCGTC ShK- 184A23 d1  SCIDTIPKSRCTAFQCKHSMKSRLSFCRKTCGTC ShK- 185 S23 d1 SCIDTIPKSRCTAFQCKHSMKFRLSFCRKTCGTC ShK- 186 F23 d1 SCIDTIPKSRCTAFQCKHSMKX^(s23)RLSFCRKTCGTC ShK- 187 X23 d1SCIDTIPKSRCTAFQCKHSMK(nitrophe) ShK- 188 RLSFCRKTCGTC Nitro- phe23 d1SCTDTIPKSRCTAFQCKHSMK(aminophe) ShK- 189 RLSFCRKTCGTC Amino- phe23 d1SCIDTIPKSRCTAFQCKHSMK(benzylphe) ShK- 190 RLSFCRKTCGTC Ben- zyl- phe23d1  SCIDTIPKSRCTAFQCKHSMKYALSFCRKTCGTC ShK- 191 A24 d1 SCIDTIPKSRCTAFQCKHSMKYELSFCRKTCGTC ShK- 192 E24 d1 SCIDTIPKSRCTAFQCKHSMKYRASFCRKTCGTC ShK- 193 A25 d1 SCIDTIPKSRCTAFQCKHSMKYRLAFCRKTCGTC ShK- 194 A26 d1 SCIDTIPKSRCTAFQCKHSMKYRLSACRKTCGTC ShK- 195 A27 d1 SCIDTIPKSRCTAFQCKHSMKYRLSXs27CRKTCGTC ShK- 196 X27 d1 SCIDTIPKSRCTAFQCKHSMKYRLSFCAKTCGTC ShK- 197 A29 d1 SCIDTIPKSRCTAFQCKHSMKYRLSFCRATCGTC ShK- 198 A30 d1 SCIDTIPKSRCTAFQCKHSMKYRLSFCRKACGTC ShK- 199 A31 d1 SCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGAC ShK- 200 A34 d2YSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-Y1 548KSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-K1 549HSCIDTIPKSRCTAFQCKHSNKYRLSFCRKTCGTC ShK-H1 550QSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-Q1 551PPRSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC PP-ShK 552MRSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC M-ShK 553GRSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC G-ShK 554YSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC ShK- 555 Y1/A22KSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC ShK- 556 K1/A22HSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC ShK- 557 H1/A22QSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC ShK- 558 Q1/A22PPRSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC PP- 559 ShK- A22MRSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC M-ShK- 560 A22GRSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC G-ShK- 561 A22RSCIDTIPASRCTAFQCKHSMAYRLSFCRKTCGTC ShK- 884 A9/A22SCIDTIPASRCTAFQCKHSMAYRLSFCRKTCGTC ShK- 885 A9/A22 d1RSCIDTIPVSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-V9 886RSCIDTIPVSRCTAFQCKHSMAYRLSFCRKTCGTC ShK- 887 V9/A22SCIDTIPVSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-V9 888 d1SCIDTIPVSRCTAFQCKHSMAYRLSFCRKTCGTC ShK- 889 V9/A22 d1RSCIDTIPESRCTAFQCKHSMAYRLSFCRKTCGTC ShK- 890 E9/A22SCIDTIPESRCTAFQCKHSMAYRLSFCRKTCGTC ShK- 891 E9/A22 d1RSCIDTIPKSACTAFQCKHSMAYRLSFCRKTCGTC ShK- 892 A11/ A22SCIDTIPKSACTAFQCKHSMAYRLSFCRKTCGTC ShK- 893 A11/22 d1RSCIDTIPKSECTAFQCKHSMAYRLSFCRKTCGTC ShK- 894 E11/ A22SCIDTIPKSECTAFQCKHSMAYRLSFCRKTCGTC ShK- 895 E11/ A22 d1RSCIDTIPKSRCTDFQCKHSMKYRLSFCRKTCGTC ShK- 896 D14RSCIDTIPKSRCTDFQCKHSMAYRLSFCRKTCGTC ShK- 897 D14/ A22SCIDTIPKSRCTDFQCKHSMKYRLSFCRKTCGTC ShK- 898 D14 d1SCIDTIPKSRCTDFQCKHSMAYRLSFCRKTCGTC ShK- 899 D14/ A22 d1RSCIDTIPKSRCTAAQCKHSMAYRLSFCRKTCGTC ShK- 900 A15A/ 22SCIDTIPKSRCTAAQCKHSMAYRLSFCRKTCGTC ShK- 901 A15/ A22 d1RSCIDTIPKSRCTAIQCKHSMKYRLSFCRKTCGTC ShK- 902 I15RSCIDTIPKSRCTAIQCKHSMAYRLSFCRKTCGTC ShK- 903 I15/ A22SCIDTIPKSRCTAIQCKHSMKYRLSFCRKTCGTC ShK- 904 I15 d1SCIDTIPKSRCTAIQCKHSMAYRLSFCRKTCGTC ShK- 905 I15/ A22 d1RSCIDTIPKSRCTAVQCKHSMKYRLSFCRKTCGTC ShK- 906 V15RSCIDTIPKSRCTAVQCKHSMAYRLSFCRKTCGTC ShK- 907 V15/ A22SCIDTIPKSRCTAVQCKHSMKYRLSFCRKTCGTC ShK- 908 V15 d1SCIDTIPKSRCTAVQCKHSMAYRLSFCRKTCGTC ShK- 909 V15/ A22 d1RSCIDTIPKSRCTAFRCKHSMKYRLSFCRKTCGTC ShK- 910 R16RSCIDTIPKSRCTAFRCKHSMAYRLSFCRKTCGTC ShK- 911 R16/ A22SCIDTIPKSRCTAFRCKHSMKYRLSFCRKTCGTC ShK- 912 R16 d1SCIDTIPKSRCTAFRCKHSMAYRLSFCRKTCGTC ShK- 913 R16/ A22 d1RSCIDTIPKSRCTAFKCKHSMKYRLSFCRKTCGTC ShK- 914 K16RSCIDTIPKSRCTAFKCKHSMAYRLSFCRKTCGTC ShK- 915 K16/ A22SCIDTIPKSRCTAFKCKHSMKYRLSFCRKTCGTC ShK- 916 K16 d1SCIDTIPKSRCTAFKCKHSMAYRLSFCRKTCGTC ShK- 917 K16/ A22 d1RSCIDTIPASECTAFQCKHSMKYRLSFCRKTCGTC ShK- 918 A9/E11RSCIDTIPASECTAFQCKHSMAYRLSFCRKTCGTC ShK- 919 A9/ E11/ A22SCIDTIPASECTAFQCKHSMKYRLSFCRKTCGTC ShK- 920 A9/E11 d1SCIDTIPASECTAFQCKHSMAYRLSFCRKTCGTC ShK- 921 A9/ E11/ A22 d1RSCIDTIPVSECTAFQCKHSMKYRLSFCRKTCGTC ShK- 922 V9/E11RSCIDTIPVSECTAFQCKHSMAYRLSFCRKTCGTC ShK- 923 V9/ E11/ A22SCIDTIPVSECTAFQCKHSMKYRLSFCRKTCGTC ShK- 924 V9/E11 d1SCIDTIPVSECTAFQCKHSMAYRLSFCRKTCGTC ShK- 925 V9/ E11/ A22 d1RSCIDTIPVSACTAFQCKHSMKYRLSFCRKTCGTC ShK- 926 V9/A11RSCIDTIPVSACTAFQCKHSMAYRLSFCRKTCGTC ShK- 927 V9/ A11/ A22SCIDTIPVSACTAFQCKHSMKYRLSFCRKTCGTC ShK- 928 V9/A11 d1SCIDTIPVSACTAFQCKHSMAYRLSFCRKTCGTC ShK- 929 V9/ A11/ A22 d1RSCIDTIPASACTAFQCKHSMKYRLSFCRKTCGTC ShK- 930 A9/A11RSCIDTIPASACTAFQCKHSMAYRLSFCRKTCGTC ShK- 931 A9/ A11/ A22SCIDTIPASACTAFQCKHSMKYRLSFCRKTCGTC ShK- 932 A9/A11 d1SCIDTIPASACTAFQCKHSMAYRLSFCRKTCGTC ShK- 933 A9/ A11/ A22 d1RSCIDTIPKSECTDIRCKHSMKYRLSFCRKTCGTC ShK- 934 E11/ D14/ I15/ R16RSCIDTIPKSECTDIRCKHSMAYRLSFCRKTCGTC ShK- 935 E11/ D14/ I15/ R16/ A22SCIDTIPKSECTDIRCKHSMKYRLSFCRKTCGTC ShK- 936 E11/ D14/ I15/ R16 d1SCIDTIPKSECTDIRCKHSMAYRLSFCRKTCGTC ShK- 937 E11/ D14/ I15// R16 A22 d1RSCIDTIPVSECTDIRCKHSMKYRLSFCRKTCGTC ShK- 938 V9/ E11/ D14/ I15/ R16RSCIDTIPVSECTDIRCKHSMAYRLSFCRKTCGTC ShK- 939 V9/ E11/ D14/ I15/ R16/ A22SCIDTIPVSECTDIRCKHSMKYRLSFCRKTCGTC ShK- 940 V9/ E11/ D14/ I15/ R16 d1SCIDTIPVSECTDIRCKHSMAYRLSFCRKTCGTC ShK- 941 V9/ E11/ D14/ I15/ R16/A22 d1 RSCIDTIPVSECTDIQCKHSMKYRLSFCRKTCGTC ShK- 942 V9/ E11/ D14/ I15RSCIDTIPVSECTDIQCKHSMAYRLSFCRKTCGTC ShK- 943 V9/ E11/ D14/ I15/A 22SCIDTIPVSECTDIQCKHSMKYRLSFCRKTCGTC ShK- 944 V9/ E11/ D14/ I15 d1SCIDTIPVSECTDIQCKHSMAYRLSFCRKTCGTC ShK- 945 V9/ E11/ D14/ I15/A 22 d1RTCKDLIPVSECTDIRCKHSMKYRLSFCRKTCGTC ShK- 946 T2/K4/ L6/V9/ E11/ D14/I15/ R16 RTCKDLIPVSECTDIRCKHSMAYRLSFCRKTCGTC ShK- 947 T2/K4/ L6/V9/ E11/D14/ I15/ R16/ A22 TCKDLIPVSECTDIRCKHSMKYRLSFCRKTCGTC ShK- 948 T2/K4/L6/V9/ E11/ D14/ I15/ R16 d1 TCKDLIPVSECTDIRCKHSMAYRLSFCRKTCGTC ShK- 949T2/K4/ L6/V9/ E11/ D14/ I15/ R16/ A22 d1 (L-Phosphotyrosine)- ShK 950AEEARSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC (L5)QSCADTIPKSRCTAAQCKHSMKYRLSFCRKTCGTC ShK Q1/A4/ 1295 A15QSCADTIPKSRCTAAQCKHSMAYRLSFCRKTCGTC ShK 1296 Q1/A4/ A15/ A22QSCADTIPKSRCTAAQCKHSM(Dap) ShK 1297 YRLSFCRKTCGTC Q1/A4/ A15/ Dap2 2QSCADTIPKSRCTAAQCKHSMKYRASFCRKTCGTC ShK 1298 Q1/A4/ A15/ A25QSCADTIPKSRCTAAQCKHSMAYRASFCRKTCGTC ShK 1299 Q1/A4/ A15/ A22/A 25QSCADTIPKSRCTAAQCKHSM(Dap) ShK 1300 YRASFCRKTCGTC Q1/A4/ A15/ Dap2 2/A25

Many peptides as described in Table 2 can be prepared as described inU.S. Pat. No. 6,077,680 issued Jun. 20, 2000 to Kem et al., which ishereby incorporated by reference in its entirety. Other peptides ofTable 2 can be prepared by techniques known in the art. For example,ShK(L5) (SEQ ID NO: 950) can be prepared as described in Beeton et al.,Targeting effector memory T cells with a selective peptide inhibitor ofKv1.3 channels for therapy of autoimmune diseases, Molec. Pharmacol.67(4): 1369-81 (2005), which is hereby incorporated by reference in itsentirety. In Table 2 and throughout the specification, X^(s15), X^(s21),X^(s22), X^(s23) and X^(s27) each independently refer to nonfunctionalamino acid residues.

TABLE 3 HmK, BgK, AeK and AsKS peptide and peptide analog sequencesShort-hand SEQ ID Sequence/structure designation NO:RTCKDLIPVSECTDIRCRTSMKYRLNLCRKTCGSC HmK 6ATCKDLIPVSECTDIRCRTSMKYRLNLCRKTCGSC HmK-A1 201STCKDLIPVSECTDIRCRTSMKYRLNLCRKTCGSC HmK-S1 202 TCKDLIPVSECTDIRCRTSMKYRLNLCRKTCGSC HmK-d1 203 SCKDLIPVSECTDIRCRTSMKYRLNLCRKTCGSC HmK-d1/S2 204 TCIDLIPVSECTDIRCRTSMKYRLNLCRKTCGSC HmK-d1/I4 205 TCKDTIPVSECTDIRCRTSMKYRLNLCRKTCGSC HmK-d1/T6 206 TCKDLIPKSECTDIRCRTSMKYRLNLCRKTCGSC HmK-d1/K9 207 TCKDLIPVSRCTDIRCRTSMKYRLNLCRKTCGSC HmK- 208 d1/R11 TCKDLIPVSECTAIRCRTSMKYRLNLCRKTCGSC HmK- 209 d1/A14 TCKDLIPVSECTDFRCRTSMKYRLNLCRKTCGSC HmK- 210 d1/F15 TCKDLIPVSECTDIQCRTSMKYRLNLCRKTCGSC HmK- 211 d1/Q16 TCKDLIPVSECTDIRCKTSMKYRLNLCRKTCGSC HmK- 212 d1/K18 TCKDLIPVSECTDIRCRHSMKYRLNLCRKTCGSC HmK- 213 d1/H19 TCKDLIPVSECTDIRCRTSMKYRLSLCRKTCGSC HmK- 214 d1/S26 TCKDLIPVSECTDIRCRTSMKYRLNFCRKTCGSC HmK- 215 d1/F27 TCKDLIPVSECTDIRCRTSMKYRLNLCRKTCGTC HmK- 216 d1/T34 TCKDLIPVSRCTDIRCRTSMKYRLNFCRKTCGSC HmK- 217 d1/R11/F27ATCKDLIPVSRCTDIRCRTSMKYRLNFCRKTCGSC HmK- 218 A1/R11/F27 TCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGSC HmK-d1/Z1 219 TCIDTIPKSRCTAFQCRTSMKYRLNFCRKTCGSC HmK-d1/Z2 220 TCADLIPASRCTAIACRTSMKYRLNFCRKTCGSC HmK-d1/Z3 221 TCADLIPASRCTAIACKHSMKYRLNFCRKTCGSC HmK-d1/Z4 222 TCADLIPASRCTAIACAHSMKYRLNFCRKTCGSC HmK-d1/Z5 223RTCKDLIPVSECTDIRCRTSMX^(h22)YRLNLCRKTCGSC HmK-X22 224ATCKDLX^(h6)PVSRCTDIRCRTSMKX^(h22)RLNX^(h26)CRKTCGSC HmK-X6, 225 22, 26VCRDWFKETACRHAKSLGNCRTSQKYRANCAKTCELC BgK 9ACRDWFKETACRHAKSLGNCRTSQKYRANCAKTCELC BgK-A1 226VCADWFKETACRHAKSLGNCRTSQKYRANCAKTCELC BgK-A3 227VCRDAFKETACRHAKSLGNCRTSQKYRANCAKTCELC BgK-A5 228VCRDWFKATACRHAKSLGNCRTSQKYRANCAKTCELC BgK-A8 229VCRDWFKEAACRHAKSLGNCRTSQKYRANCAKTCELC BgK-A9 230VCRDWFKETACAHAKSLGNCRTSQKYRANCAKTCELC BgK-A12 231VCRDWFKETACRHAASLGNCRTSQKYRANCAKTCELC BgK-A15 232VCRDWFKETACRHAKALGNCRTSQKYRANCAKTCELC BgK-A16 233VCRDWFKETACRHAKSAGNCRTSQKYRANCAKTCELC BgK-A17 234VCRDWFKETACRHAKSLGNCATSQKYRANCAKTCELC BgK-A21 235VCRDWFKETACRHAKSLGNCRASQKYRANCAKTCELC BgK-A22 236VCRDWFKETACRHAKSLGNCRTSQKYAANCAKTCELC BgK-A27 237VCRDWFKETACRHAKSLGNCRTSQKYRANCAATCELC BgK-A32 238VCRDWFKETACRHAKSLGNCRTSQKYRANCAKACELC BgK-A33 239VCRDWFKETACRHAKSLGNCRTSQKYRANCAKTCALC BgK-A35 240VCRDWFKETACRHAKSLGNCRTSQKYRANCAKTCEAC BgK-A37 241GCKDNFSANTCKHVKANNNCGSQKYATNCAKTCGKC AeK 7ACKDNFAAATCKHVKENKNCGSQKYATNCAKTCGKC AsKS 8

In Table 3 and throughout the specification, X^(h6), X^(h22), X^(h26)are each independently nonfunctional residues.

TABLE 4 MgTx peptide and MgTx peptide analog sequences Short- hand SEQdesig- ID Sequence/structure nation NO:TIINVKCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPH MgTx 28TIINVACTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPH MgTx- 242 A6TIINVSCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPH MgTx- 243 S6TIINVKCTSPAQCLPPCKAQEGQSAGAKCMNGKCKCYPH MgTx- 244 A11TIINVKCTSPKQCLPPCAAQFGQSAGAKCMNGKCKCYPH MgTx- 245 A18TIINVKCTSPKQCLPPCKAQFGQSAGAACMNGKCKCYPH MgTx- 246 A28TIINVKCTSPKQCLPPCKAQFGQSAGAKCMNGACKCYPH MgTx- 247 A33TIINVKCTSPKQCLPPCKAQFGQSAGAKCMNGKCACYPH MgTx- 248 A35TIINVKCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPN MgTx- 249 H39NTIINVACTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPN MgTx- 250 A6/ H39NTIINVSCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYS MgTx- 251 S6/38/ d39TIITISCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPH MgTx- 252 T4/I5/ S6   TISCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPH MgTx- 253 d3/T4/ I5/S6   TISCTSPKQCLPPCKAQFGQSAGAKCMNGKGKCFGR MgTx- 254 Pi2   NVACTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPH MgTx- 255 d3/A6QFTNVSCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYS MgTx- 256 ChTxQFTDVDCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYQ MgTx- 257 IbTx IINVSCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPH MgTx- 258 Z1 IITISCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPH MgTx- 259 Z2GVIINVSCTSPKQCLPPCKAQEGQSAGAKCMNGKCKGYPH MgTx- 260 Z3

Many peptides as described in Table 4 can be prepared as described in WO95/03065, published Feb. 2, 1995, for which the applicant is Merck &Co., Inc. That application corresponds to U.S. Ser. No. 07/096,942,filed 22 Jul. 1993, which is hereby incorporated by reference in itsentirety.

TABLE 5 AgTx2 peptide and AgTx2 peptide analog sequences Short- hand SEQdesigna- ID Sequence/structure tion NO:GVPINVSCTGSPQCIKPCKDAGMRFGKCMNRKCHCTPK AgTx2 23GVPIAVSCTGSPQCIKPCKDAGMRFGKCMNRKCHCTPK AgTx2-A5 261GVPINVSCTGSPQCIAPCKDAGMRFGKCMNRKCHCTPK AgTx2- 262 A16GVPINVSCTGSPQCIKPCADAGMRFGKCMNRKCHCTPK AgTx2- 263 A19GVPINVSCTGSPQCIKPCKDAGMAFGKCMNRKCHCTPK AgTx2- 264 A24GVPINVSCTGSPQCIKPCKDAGMRFGACMNRKCHCTPK AgTx2- 265 A27GVPINVSCTGSPQCIKPCKDAGMRFGKCMNAKCHCTPK AgTx2- 266 A31GVPINVSCTGSPQCIKPCKDAGMRFGKCMNRACHCTPK AgTx2- 267 A32GVPINVSCTGSPQCIKPCKDAGMRFGKCMNRKCHCTPA AgTx2- 268 A38GVPIAVSCTGSPQCIKPCKDAGMRFGKCMNRKCHCTPA AgTx2- 269 A5/A38GVPINVSCTGSPQCIKPCKDAGMRFGKCMNGKCHCTPK AgTx2- 270 G31GVPIIVSCKGSRQCIKPCKDAGMRFGKCMNGKCHCTPK AgTx2- 271 OSK_z1GVPIIVSCKISRQCIKPCKDAGMRFGKCMNGKCHCTPK AgTx2- 272 OSK_z2GVPIIVKCKGSRQCIKPCKDAGMRFGKCMNGKCHCTPK AgTx2- 273 OSK_z3GVPIIVKCKISRQCIKPCKDAGMRFGKCMNGKCHCTPK AgTx2- 274 OSK_z4GVPIIVKCKISRQCIKPCKDAGMRFGKCMNGKCHCTPK AgTx2- 275 OSK_z5

TABLE 6 Heteromitrus spinnifer (HsTx1) peptide andHsTx1 peptide analog sequences Short- hand SEQ desig- IDSequence/structure nation NO: ASCRTPKDCADPCRKETGCPYGKCMNRKCKCNRC HsTx161 ASCXTPKDCADPCRKETGCPYGKCMNRKCKCNRC HsTx1-X4 276ASCATPKDCADPCRKETGCPYGKCMNRKCKCNRC HsTx1-A4 277ASCRTPXDCADPCRKETGCPYGKCMNRKCKCNRC HsTx1-X7 278ASCRTPADCADPCRKETGCPYGKCMNRKCKCNRC HsTx1-A7 279ASCRTPKDCADPCXKETGCPYGKCMNRKCKCNRC HsTx1-X14 280ASCRTPKDCADPCAKETGCPYGKCMNRKCKCNRC HsTx1-A14 281ASCRTPKDCADPCRXETGCPYGKCMNRKCKCNRC HsTx1-X15 282ASCRTPKDCADPCRAETGCPYGKCMNRKCKCNRC HsTx1-A15 283ASCRTPKDCADPCRKETGCPYGXGMNRKCKCNRC HsTx1-X23 284ASCRTPKDCADPCRKETGCPYGACMNRKCKCNRC HsTx1-A23 285ASCRTPKDCADPCRKETGCPYGKCMNXKCKCNRC HsTx1-X27 286ASCRTPKDCADPCRKETGCPYGKCMNAKCKCNRC HsTx1-A27 287ASCRTPKDCADPCRKETGCPYGKCMNRXCKCNRC HsTx1-X28 288ASCRTPKDCADPCRKETGCPYGKCMNRACKCNRC HsTx1-A28 289ASCRTPKDCADPCRKETGCPYGKCMNRKCXCNRC HsTx1-X30 290ASCRTPKDCADPCRKETGCPYGKCMNRKCACNRC HsTx1-A30 291ASCRTPKDCADPCRKETGCPYGKCMNRKCKCNXC HsTx1-X33 292ASCRTPKDCADPCRKETGCPYGKCMNRKCKCNAC HsTx2-A33 293

Peptides as described in Table 5 can be prepared as described in U.S.Pat. No. 6,689,749, issued Feb. 10, 2004 to Lebrun et al., which ishereby incorporated by reference in its entirety.

TABLE 7 Orthochirus scrobiculosus (OSK1) peptide and OSK1 peptide analogsequences SEQ Short-hand ID Sequence/structure designation NO:GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK OSK1 25GVIINVSCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK OSK1-S7 1303GVIINVKCKISRQCLKPCKKAGMRFGKCMNGKCHCTPK OSK1-K16 294GVIINVKCKISRQCLEPCKDAGMRFGKCMNGKCHCTPK OSK1-D20 295GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK OSK1-K16, D20 296GVIINVSCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK OSK1-S7, K16, D20 1308GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTPK OSK1-P12, K16, D20 297GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCYPK OSK1-K16, D20, Y36 298Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTPK Ac-OSK1-P12, 562 K16, D20GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ OSK1-P12, K16, 563 D20-NH₂AC-GVIINVKCKISPQCLKPCKDACMRFGKCMNGKCHCTPK-NH₂ Ac-OSK1-P12, 564K16, D20-NH₂ GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCYPK-NH₂ OSK1-K16, D20,565 Y36-NH₂ Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCYPK Ac-OSK1-K16, 566D20, Y36 Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCYPK-NH₂ Ac-OSK1-K16, D20,567 Y36-NH₂ GVIINVKCKISRQCLKPCKKAGMRFGKCMNGKCHCTPK-NH₂ OSK1-K16-NH₂ 568Ac-GVIINVKCKISRQCLKPCKKAGMRFGKCMNGKCHCTPK Ac-OSK1-K16 569Ac-GVIINVKCKISRQCLKPCKKAGMRFGKCMNGKCHCTPK-NH₂ Ac-OSK1-K16-NH₂ 570Ac-GVIINVKCKISRQCLEPCKDAGMRFGKCMNGKCHCTPK Ac-OSK1-D20 571GVIINVKCKISRQCLEPCKDAGMRFGKCMNGKCHCTPK-NH₂ OSK1-D20-NH₂ 572Ac-GVIINVKCKISRQCLEPCKDAGMRFGKCMNGKCHCTPK-NH₂ Ac-OSK1-D20-NH₂ 573GVIINVKCKISRQCLEPCKKAGMRFCKCMNGKCHCTPK-NH₂ OSK1-NH₂ 574Ac-GVIINVKCKISRQCLEPGKKAGMRFGKCMNGKCHCTPK Ac-OSK1 575Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-NH₂ Ac-OSK1-NH₂ 576GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ OSK1-K16, D20-NH₂ 577Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK Ac-OSK1-K16, 578 D20Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ Ac-OSK1-K16, D20- 579 NH₂VIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK Δ1-OSK1 580Ac-VIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK Ac-Δ1-OSK1 581VIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-NH₂ Δ1-OSK1-NH₂ 582Ac-VIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-NH₂ Ac-Δ1-OSK1-NH₂ 583GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK OSK1-A34 584Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK Ac-OSK1-A34 585GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK-NH₂ OSK1-A34-NH₂ 586Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPKNH₂ AC-OSK1-A34-NH₂ 587VIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK Δ1-OSK1-K16, D20 588Ac-VIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK Ac-Δ1-OSK1-K16, 589 D20VIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ Δ1-OSK1-K16, D20- 590 NH₂Ac-VIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ Ac-Δ1-OSK1-K16, 591 D20-NH₂NVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK (Δ1-4)-OSK1-K16, 592 D20Ac-NVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK Ac-(Δ1-4)-OSK1- 593 K16, D20NVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ (Δ1-4)-OSK1-K16, 594 D20-NH₂Ac-NVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ Ac-(Δ1-4)-OSK1- 595K16, D20-NH₂ KCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK (Δ1-6)-OSK1-K16, 596 D20Ac-KCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK Ac-(Δ1-6)-OSK1- 597 K16, D20KCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ (Δ1-6)-OSK1-K16, 598 D20-NH₂Ac-KCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ Ac-(Δ1-6)-OSK1- 599 K16, D20-NH₂CKISRQCLKPCKDAGMRFGKCMNGKCHCTPK (Δ1-7)-OSK1-K16, 600 D20Ac-CKISRQCLKPCKDAGMRFGKCMNGKCHCTPK Ac-(Δ1-7)-OSK1- 601 K16, D20CKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ (Δ1-7)-OSK1-K16, 602 D20-NH₂Ac-CKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ Ac-(Δ1-7)-OSK1- 603 K16, D20-NH₂GVIINVKCKISRQCLKPCKDAGMRNGKCMNGKCHCTPK OSK1-K16, D20, 604 N25GVIINVKCKISRQCLKPCKDAGMRNGKCMNGKCHCTPK-NH₂ OSK1-K16, D20, 605 N25-NH₂Ac-GVIINVKCKISRQCLKPCKDAGMRNGKCMNGKCHCTPK Ac-OSK1-K16, 606 D20, N25Ac-GVIINVKCKISRQCLKPCKDAGMRNGKCMNGKCHCTPK-NH₂ Ac-OSK1-K16, D20, 607N25-NH₂ GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK OSK1-K16, D20, 608 R31GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHGTPK-NH₂ OSK1-K16, D20, 609 R31-NH₂Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK Ac-OSK1-K16, 610 D20, R31Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK-NH₂ Ac-OSK1-K16, D20, 611R31-NH₂ GVINVKCKISKQCLKPCRDAGMRFGKCMNGKCHCTPK OSK1-K12, K16, 612R19, D20 Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCMNGKCHCTPK Ac-OSK1-K12, K16, 613R19, D20 GVIINVKCKISKQCLKPCRDAGMRFGKCMNGKCHCTPK-NH₂ OSK1-K12, K16, 614R19, D20-NH₂ Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCMNGKCHCTPK-NH₂Ac-OSK1-K12, K16, 615 R19, D20-NH₂ TIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPKΔ1-OSK1-T2, K16, 616 D20 Ac-TIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPKAc-Δ1-OSK1-T2, 617 K16, D20 TIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂Δ1-OSK1-T2, K16, 618 D20-NH₂Ac-TIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ Ac-A1-OSK1-T2, 619K16, D20-NH₂ GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK OSK1-K3 620Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK Ac-OSK1-K3 621GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-NH₂ OSK1-K3-NH₂ 622Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-NH₂ Ac-OSK1-K3-NH₂ 623GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK OSK1-K3, A34 624GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK OSK1-K3, K16, D20 625GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCACTPK OSK1-K3, K16, D20, 626 A34Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK Ac-OSK1-K3, A34 627GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK-NH₂ OSK1-K3, A34-NH₂ 628Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK-NH₂ Ac-OSK1-K3, A34- 629 NH₂Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCACTPK Ac-OSK1-K3, K16, 630 D20, A34GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCACTPK-NH₂ OSK1-K3, K16, D20, 631A34-NH₂ Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCACTPK-NH₂ Ac-OSK1-K3, K16,632 D20, A34-NH₂ Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPKAc-OSK1-K3, K16, 633 D20 GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂OSK1-K3, K16, D20- 634 NH₂ Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂Ac-OSK1-K3, K16, 635 D20-NH₂ GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTΔ36-38-OSK1-K16, 636 D20 GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHCTPKOSK1-O16, D20 980 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKGHCTPK OSK1-hLys981 16, D20 GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCTPK OSK1-hArg 98216, D20 GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCTPK OSK1-Cit 16, D20 983GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCTPK OSK1-hCit 984 16, D20GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCTPK OSK1-Dpr 16, D20 985GVIINVKCKISRQCL[Dab]PCKDACMRFGKCMNGKCHCTPK OSK1-Dab 16, D20 986GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHCYPK OSK1-O16, D20, Y36 987GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHCYPK OSK1-hLys 988 16, D20, Y36GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCYPK OSK1-hArg 989 16, D20, Y36GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCYPK OSK1-Cit 990 16, D20, Y36GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCYPK OSK1-hCit 991 16, D20, Y36GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCYPK OSK1-Dpr 992 16, D20, Y36GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCHCYPK OSK1-Dab 993 16, D20, Y36GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACYPK OSK1- 994 K16, D20, A34, Y36GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCGCYPK OSK1- 995 K16, D20, G34, Y36GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACFPK OSK1- 996 K16, D20, A34, F36GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACWPK OSK1- 997 K16, D20, A34, W36GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCACYPK OSK1- 998 K16, E20, A34, Y36GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACTPK OSK1-016, D20, A34 999GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCACTPK OSK1-hLys 1000 16, D20, A34GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCACTPK OSK1-hArg 1001 16, D20, A34GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCACTPK OSK1-Cit 1002 16, D20, A34GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCTPK OSK1-hCit 1003 16, D20, A34GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCACTPK OSK1-Dpr 1004 16, D20, A34GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCACTPK OSK1-Dab 1005 16, D20, A34GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHC Δ36-38, OSK1- 1006 O16, D20,GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHC Δ36-38, OSK1- 1007 hLys 16, D20GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHC Δ36-38, OSK1- 1008 hArg 16, D20GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHC Δ36-38, OSK1-Cit 1009 16, D20GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHC Δ36-38, OSK1- 1010 hCit 16, D20GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHC Δ36-38, OSK1-Dpr 1011 16, D20GVIINVKCKTSRQCL[Dab]PCKDAGMRFGKCMNGKCHC Δ36-38, OSK1- 1012 Dab16, D20GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCAC Δ36-38, OSK1- 1013 O16, D20, A34GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCAC Δ36-38, OSK1- 1014hLys 16, D20, A34 GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCAC Δ36-38, OSK1-1015 hArg 16, D20, A34 GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCACΔ36-38, OSK1-Cit 1016 16, D20, A34GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHC Δ36-38, OSK1- 1017hCit 16, D20, A34 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCACΔ36-38, OSK1-Dpr 1018 16, D20, A34GVIINVKCKISRQCL[Dab]PCKDAGMRFCKCMNGKCAC Δ36-38, OSK1-Dab 101916, D20, A34 GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCGCYGG OSK1- 1020K16, D20, G34, Y36, G37, G38 GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHCYGGOSK1- 1021 O16, D20, Y36, G37, G38GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHCYGG OSK1-hLys 102216, D20, Y36, G37, G38 GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCYGGOSK1-hArg 1023 16, D20, Y36, G37, G38GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCYGG OSK1-Cit 102416, D20, Y36, G37, G38 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCYGGOSK1-hCit 1025 16, D20, Y36, G37,  G38GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCYGG OSK1-Dpr 102616, D20, Y36, G37,  G38 GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACYGG OSK1-1027 K16, D20, A34, Y36,  G37, G38GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACYGG OSK1- 1028 O16, D20, A34, Y36, G37, G38 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCACYGG OSK1-hLys 102916, D20, A34, Y36,  G37, G38 GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCACYGGOSK1-hArg 1030 16, D20, A34, Y36,  G37, G38GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCACYGG OSK1-Cit 103116, D20, A34, Y36,  G37, G38 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCYGGOSK1-hCit 1032 16, D20, A34, Y3, G37, G38GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCACYGG OSK1-Dpr 103316, D20, A34, Y36,  G37, G38 GVIINVKCKISRQCL[Dab]PCKDAGMREGKCMNGKCACYGGOSK1-Dab 1034 16, D20, A34, Y36,  G37, G38GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACYG Δ38, OSK1- 1035K16, D20, A34, Y36,  G37 GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHCGGG OSK1-1036 O16, D20, G36-38 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHCGGGOSK1-hLys 1037 16, D20, G36-38GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCGGG OSK1-hArg 103816, D20, G36-38 GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCGGG OSK1-Cit 103916, D20, G36-38 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCGGG OSK1-hCit1040 16, D20, G36-38 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCGGG OSK1-Dpr1041 16, D20, G36-38 GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACFGG OSK1- 1042K16, D20, A34, F36,  G37, G38 GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACGGGOSK1- 1043 O16, D20, A34, G36- 38GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCACGGG OSK1-hLys 104416, D20, A34, G36-38 GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCACGGGOSK1-hArg 1045 16, D20, A34, G36-38GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCACGGG OSK1-Cit 104616, D20, A34, G36-38 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCACGGGOSK1-hCit 1047 16, D20, A34, G36-38GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCACGGG OSK1-Dpr 104816, D20, A34, G36-38 GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCACGGG OSK1-Dab1049 16, D20, A34, G36-38 GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACGGΔ38, OSK1- 1050 K16, D20, A34, G36- 37GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACYG Δ38, OSK1- 1051K16, D20, A35, Y36,  G37 GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACGGA38, OSK1- 1052 O16, D20, A35, Y36, G37GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCTPK OSK1-hLys 1053 16, E20GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCTPK OSK1-hArg 1054 16, E20GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCTPK OSK1-Cit 16, E20 1055GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCTPK OSK1-hCit 1056 16, E20GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCTPK OSK1-Dpr 16, E20 1057GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCHCTPK OSK1-Dab 16, E20 1058GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHCYPK OSK1-O16, E20, Y36 1059GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCYPK OSK1-hLys 1060 16, E20, Y36GVIINVKCKISRQCL[hArg]PCKEAGMRPGKCMNGKCHCYPK OSK1-hArg 1061 16, E20, Y36GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCYPK OSK1-Cit 1062 16, E20, Y36GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCYPK OSK1-hCit 1063 16, E20, Y36GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCYPK OSK1-Dpr 1064 16, E20, Y36GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCHCYPK OSK1-Dab 1065 16, E20, Y36GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCACTPK OSK1-O16, E20, A34 1066GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCACTPK OSK1-hLys 1067 16, E20, A34GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCACTPK OSK1-hArg 1068 16, E20, A34GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCACTPK OSK1-Cit 1069 16, E20, A34GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCTPK OSK1-hCit 1070 16, E20, A34GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCACTPK OSK1-Dpr 1071 16, E20, A34GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCACTPK OSK1-Dab 1072 16, E20, A34GVIINVKCKISRQCLOPCKEAGMRPGKCMNGKCHC Δ36-38, OSK1- 1073 O16, E20,GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHC Δ36-38, OSK1- 1074 hLys 16, E20GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHC Δ36-38, OSK1- 1075 hArg 16, E20GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHC Δ36-38, OSK1-Cit 1076 16, E20GVIINVKCKISRQCL[hCit]PCKEAGMREGKCMNGKCHC Δ36-38, OSK1- 1077 hCit 16, E20GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHC Δ36-38, OSK1-Dpr 1078 16, E20GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCAC Δ36-38, OSK1- 1079 O16, E20, A34GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCAC Δ36-38, OSK1- 1080hLys 16, E20, A34 GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCAC Δ36-38 OSK1-1081 hArg 16, E20, A34 GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCACΔ36-38, OSK1-Cit 1082 16, E20, A34GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHC Δ36-38, OSK1- 1083hCit 16, E20, A34 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCACΔ36-38, OSK1-Dpr 1084 16, E20, A34GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCAC Δ36-38, OSK1-Dab 108516, E20, A34 GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCHCYGG OSK1- 1086K16, E20, Y36, G37,  G38 GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHCYGG OSK1-1087 O16, E20, Y36, G37,  G38 GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCHCYGΔ38 OSK1- 1088 K16, E20, Y36, G37 GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCACYGΔ38 OSK1- 1089 K16, E20, A34, Y36, G37GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCYGG OSK1-hLys 109016, E20, Y36, G37,  G38 GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCYGGOSK1-hArg 1091 16, E20, Y36, G37,  G38GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCYGG OSK1-Cit 109216, E20, Y36, G37,  G38 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCYGGΔ37-38, OSK1- 1093 ΔCit 16, E20, Y36, G37,  G38GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCYGG OSK1-Dpr 109416, E20, Y36, G37,  G38 GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCHCYGGOSK1-Dab 1095 16, E20, Y36, G37,  G38GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCACYG Δ38, OSK1- 1096K16, E20, A34, Y36,  G37 GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCACYGG OSK1-1097 O16, E20, A34, Y36,  G37, G38GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCACYGG OSK1-hLys 109816, E20, A34, Y36,  G37, G38 GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCACYGGOSK1-hArg 1099 16, E20, A34, Y36,  G37, G38GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCACYGG OSK1-Cit 110016, E20, A34, Y36,  G37, G38 GVIINVKCKISRQCL[hCit]PCKEAGMREGKCMNGKCHCYGGOSK1-hCit 1101 16, E20, A34, Y3, G37, G38GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCACYGG OSK1-Dpr 110216, E20, A34, Y36,  G37, G38 GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCACYGGOSK1-Dab 1103 16, E20, A34, Y36,  G37, G38GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCACFGG OSK1- 1104 K16, D20, A34, F36, G37, G38 GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHCGGG OSK1- 1105O16, E20, G36-38 GVIINVKCKISRQCL[hLys]PCKEAGMREGKCMNGKCHCGGG OSK1-hLys1106 16, E20, G36-38 GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCGGGOSK1-hArg 1107 16, E20, G36-38GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCGGG OSK1-Cit 1108 16, E20, G36-38GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCGGG OSK1-hCit 110916, E20, G36-38 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCGGG OSK1-Dpr 111016, E20, G36-38 GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCACGGG OSK1- 1111O16, E20, A34, G36- 38 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCACGGGOSK1-hLys 1112 16, E20, A34, G36-38GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCACGGG OSK1-hArg 111316, E20, A34, G36-38 GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCACGGG OSK1-Cit1114 16, E20, A34, G36-38 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCACTPOSK1-hCit 1115 16, E20, A34, G36-38GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCACTP OSK1-Dpr 111616, E20, A34, G36-38 GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCACTP OSK1-Dab1117 16, E20, A34, G36-38 GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHCTPK-NH₂OSK1-O16, D20- 1118 amideGVIINVKCKISRQCL[hLys]PCKDAGMRFCKCMNGKCHCTPK-NH₂ OSK1-hLys 111916, D20-amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCTPK-NH₂ OSK1-hArg1120 16, D20-amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCTPK-NH₂OSK1-Cit 1121 16, D20-amideGVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCTPK-NH₂ OSK1-hCit 112216, D20-amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCTPK-NH₂ OSK1-Dpr1123 16, D20-amide GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCHCTPK-NH₂OSK1-Dab 16, D20 1124 GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHCYPK-NH₂ OSK1-1125 O16, D20, Y36- amideGVIINVKCKISRQCL[hLys]PCKDAGMRFCKCMNGKCHCYPK-NH₂ OSK1-hLys 112616, D20, Y36- amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCYPK-NH₂OSK1-hArg 1127 16, D20,Y36- amideGVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCYPK-NH₂ OSK1-Cit 112816, D20, Y36- amide GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCYPK-NH₂OSK1-hCit 1129 16, D20, Y36- amideGVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCYPK-NH₂ OSK1-Dpr 113016, D20, Y36- amide GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCHCYPK-NH₂OSK1-Dab 1131 16, D20, Y36- amideGVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACTPK-NH₂ OSK1- 1132 O16, D20, A34-amide GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCACTPK-NH₂ OSK1-hLys 113316, D20, A34- amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCACTPK-NH₂OSK1-hArg 1134 16, D20, A34- amideGVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCACTPK-NH₂ OSK1-Cit 113516, D20, A34- amide GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCACTPK-NH₂OSK1-hCit 1136 16, D20, A34- amideGVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCACTPK-NH₂ OSK1-Dpr 113716, D20, A34- amide GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCACTPK-NH₂OSK1-Dab 1138 16, D20, A34- amideGVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHC-NH₂ Δ36-38, OSK1- 1139 O16, D20, -amide GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHC-NH₂ Δ36-38, OSK1- 1140hLys 16, D20- amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHC-NH₂Δ36-38, OSK1- 1141 hArg 16, D20- amideGVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHC-NH₂ Δ36-38, OSK1-Cit 114216, D20-amide GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHC-NH₂ Δ36-38, OSK1-1143 hCit 16, D20- amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHC-NH₂Δ36-38, OSK1-Dpr 1144 16, D20-amideGVIINVKCKISRQCLOPCKDAGMRFGKGMNGKCAC-NH₂ Δ36-38, OSK1- 1145O16, D20, A34- amide GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCAC-NH₂Δ36-38, OSK1- 1146 hLys 16, D20, A34- amideGVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCAC-NH₂ Δ36-38, OSK1- 1147hArg 16, D20, A34- amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCAC-NH₂Δ36-38, OSK1-Cit 1148 16, D20, A34- amideGVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHC-NH₂ Δ36-38, OSK1- 1149hCit 16, D20, A34 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCAC-NH₂Δ36-38, OSK1-Dpr 1150 16, D20, A34- amideGVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCAC-NH₂ Δ36-38, OSK1-Dab 115116, D20, A34- amide GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCYGG-NH₂ OSK1-1152 O16, D20, Y36, G37,  G38-amideGVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHCYGG-NH₂ OSK1- 1153O16, D20, Y36, G37,  G38 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHCYGG-NH₂OSK1-hLys 1154 16, D20, Y36, G37,  G38-amideGVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCYGG-NH₂ OSK1-hArg 115516, D20, Y36, G37,  G38-amideGVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCYGG-NH₂ OSK1-Cit 115616, D20, Y36, G37,  G38-amideGVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCYGG-NH₂ OSK1- 1157hCit 16, D20, Y36,  G37, G38-amideGVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCYGG-NH₂ OSK1-Dpr 115816, D20, Y36, G37,  G38-amide GVIINVKCKISRQCLKPCKDAGMRPGKCMNGKCHCFGG-NH₂OSK1- 1159 K16, D20, F36, G37,  G38-amideGVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCYG-NH₂ Δ38-OSK1- 1160K16, D20, Y36, G37- amide GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACYG-NH₂Δ38-OSK1- 1161 K16, D20, A34, Y36, G37-amideGVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACYGG-NH₂ OSK1- 1162O16, D20, A34, Y36,  G37, G38-amideGVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCACYGG-NH₂ OSK1-hLys 116316, D20, A34, Y36,  G37, G38-amideGVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCACYGG-NH₂ OSK1-hArg 116416, D20, A34, Y36,  G37, G38-amideGVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCACYGG-NH₂ OSK1-Cit 116516, D20, A34, Y36,  G37, G38GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCACYGG-NH₂ OSK1- 1166hCit 16, D20, A34,  Y3, G37, G38-amideGVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCACYGG-NH₂ OSK1-Dpr 116716, D20, A34, Y36,  G37, G38-amideGVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCACYGG-NH₂ OSK1-Dab 116816, D20, A34, Y36,  G37, G38-amideGVIINVKCKISRQCLKPGKDAGMRFGKCMNGKCACYGG-NH₂ OSK1- 1169K16, D20, A34, Y36,  G37, G38-amideGVIINVKCKISRQCLOPCKDAGMRFGKGMNGKCHCGGG-NH₂ OSK1- 1170 O16, D20, G36-38-amide GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHCGGG-NH₂ OSK1-hLys 117116, D20, G36-38- amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCGGG-NH₂OSK1-hArg 1172 16, D20, G36-38- amideGVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCGGG-NH₂ OSK1-Cit 117316, D20, G36-38- amide GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCGGG-NH₂OSK1- 1174 hCit 16, D20, G36- 38-amideGVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCGGG-NH₂ OSK1-Dpr 117516, D20, G36-38- amide GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACGGG-NH₂ OSK1-1176 K16, D20, A34, G36- 38-amideGVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACFGG-NH₂ OSK1- 1177O16, D20, A34, F36, G37-38-amideGVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACGGG-NH₂ OSK1- 1178O16, D20, A34, G36- 38-amideGVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCACGGG-NH₂ OSK1-hLys 117916, D20, A34, G36- 38-amideGVIINVKCKTSRQCL[hArg]PCKDAGMRFGKCMNGKCACGGG-NH₂ OSK1-hArg 118016, D20, A34, G36- 38-amideGVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCACGGG-NH₂ OSK1-Cit 118116, D20, A34, G36- 38-amideGVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCACGGG-NH₂ OSK1- 1182hCit 16, D20, A34,  G36-38-amideGVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCACGGG-NH₂ OSK1-Dpr 118316, D20, A34, G36- 38-amideGVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCACGGG-NH₂ OSK1-Dab 118416, D20, A34, G36- 38-amide GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHCTPK-NH₂OSK1-016, E20- 1185 amideGVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCTPK-NH₂ OSK1-hLys 118616, E20-amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCTPK-NH₂ OSK1-hArg1187 16, E20-amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCTPK-NH₂OSK1-Cit 1188 16, E20-amideGVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCTPK-NH₂ OSK1- 1189 hCit 16, E20-amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCTPK-NH₂ OSK1-Dpr 119016, E20-amide GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCHCTPK-NH₂ OSK1-Dab1191 16, E20-amide GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHCYPK-NH₂ OSK1- 1192O16, E20, Y36- amide GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCYPK-NH₂OSK1-hLys 1193 16, E20, Y36- amideGVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCYPK-NH₂ OSK1-hArg 119416, E20, Y36- amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCYPK-NH₂OSK1-Cit 1195 16, E20, Y36- amideGVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCYPK-NH₂ OSK1- 1196hCit 16, E20, Y36- amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCYPK-NH₂OSK1-Dpr 1197 16, E20, Y36- amideGVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCHCYPK-NH₂ OSK1-Dab 119816, E20, Y36- amide GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCACTPK-NH₂ OSK1-1199 O16, E20, A34- amideGVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCACTPK-NH₂ OSK1-hLys 120016, E20, A34- amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCACTPK-NH₂OSK1-hArg 1201 16, E20, A34- amideGVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCACTPK-NH₂ OSK1-Cit 120216, E20, A34- amide GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCACTPK-NH₂OSK1- 1203 hCit 16, E20, A34- amideGVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCACTPK-NH₂ OSK1-Dpr 120416, E20, A34- amide GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCACTPK-NH₂OSK1-Dab 1205 16, E20, A34- amideGVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHC-NH₂ Δ36-38, OSK1- 1206 O16, E20, -amide GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHC-NH₂ Δ36-38, OSK1- 1207hLys 16, E20- amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHC-NH₂Δ36-38, OSK1- 1208 hArg 16, E20- amideGVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHC-NH₂ Δ36-38, OSK1-Cit 120916, E20-amide GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHC-NH₂ Δ36-38, OSK1-1210 hCit 16, E20- amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHC-NH₂Δ36-38, OSK1-Dpr 1211 16, E20-amideGVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCAC-NH₂ Δ36-38, OSK1- 1212O16, E20, A34- amide GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCAC-NH₂Δ36-38, OSK1- 1213 hLys 16, E20, A34- amideGVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCAC-NH₂ Δ36-38, OSK1- 1214hArg16, E20, A34- amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCAC-NH₂Δ36-38, OSK1-Cit 1215 16, E20, A34- amideGVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHC-NH₂ Δ36-38, OSK1- 1216hCit 16, E20, A34- amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCAC-NH₂Δ36-38, OSK1-Dpr 1217 16, E20, A34- amideGVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCAC-NH₂ Δ36-38, OSK1-Dab 121816, E20, A34- amide GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCHCWGG-NH₂ OSK1-1219 O16, E20, W36, G37, G38-amideGVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHCYGG-NH₂ OSK1- 1220O16, E20, Y36, G37,  G38-amideGVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCYGG-NH₂ OSK1-hLys 122116, E20, Y36, G37,  G38-amideGVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCYGG-NH₂ OSK1-hArg 122216, E20, Y36, G37,  G38-amideGVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCYGG-NH₂ OSK1-Cit 122316, E20, Y36, G37,  G38-amideGVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCYGG-NH₂ OSK1-hCit 122416, E20, Y36, G37,  G38-amideGVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCYGG-NH₂ OSK1-Dpr 122516, E20, Y36, G37,  G38-amideGVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCHCYGG-NH₂ OSK1-Dpr 122616, E20, Y36, G37,  G38-amide GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCACYGG-NH₂OSK1- 1227 K16, E20, A34, Y36,  G37, G38-amideGVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCACYGG-NH₂ OSK1- 1228O16, E20, A34, Y36,  G37, G38-amideGVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCACYGG-NH₂ OSK1-hLys 122916, E20, A34, Y36,  G37, G38-amideGVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCACYGG-NH₂ OSK1-hArg 123016, E20, A34, Y36,  G37, G38-amideGVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCACYGG-NH₂ OSK1-Cit 123116, E20, A34, Y36,  G37, G38-amideGVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCYGG-NH₂ OSK1-hCit 123216, E20, A34, Y3, G37, G38-amideGVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCACYGG-NH₂ OSK1-Dpr 123316, E20, A34, Y36,  G37, G38-amideGVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCACYGG-NH₂ OSK1-Dab 123416, E20, A34, Y36,  G37, G38-amideGVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHCGGG-NH₂ OSK1- 1235 O16, E20, G36-38-amide GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCGGG-NH₂ OSK1-hLys 123616, E20, G36-38- amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCGGG-NH₂OSK1-hArg 1237 16, E20, G36-38- amideGVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCGGG-NH₂ OSK1-Cit 123816, E20, G36-38- amide GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCGGG-NH₂OSK1-hCit 1239 16, E20, G36-38- amideGVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCGGG-NH₂ OSK1-Dpr 124016, E20, G36-38- amide GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCACGGG-NH₂ OSK1-1241 O16, E20, A34, G36- 38-amideGVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCACGGG-NH₂ OSK1-hLys 124216, E20, A34, G36- 38-amideGVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCACGGG-NH₂ OSK1-hArg 124316, E20, A34, G36- 38-amideGVIINVKCKISRQCL[Cit]PCKEAGMRECKCMNGKCACGGG-NH₂ OSK1-Cit 124416, E20, A34, G36- 38-amideGVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCACTP-NH₂ Δ38 OSK1-hCit 124516, E20, A34- amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCACGGG-NH₂OSK1-Dpr 1246 16, E20, A34, G36- 38-amideGVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCACGGG-NH₂ OSK1-Dab 124716, E20, A34, G36- 38-amideGVIINVKCKISRQCLKPCK[Cpa]AGMRFGKCMNGKCACYGG-NH₂ OSK1-K 124816, CPA20, A34, Y36, G37, G38-amideGVIINVKCKISRQCLKPCK[Cpa]AGMRFGKCMNGKCACGGG-NH₂ OSK1-K 124916, CPA20, A34, G36- 38-amideGVIINVKCKISRQCLKPCK[Cpa]AGMRFGKCMNGKCACY-NH₂ A37-380SK1-K 125016, CPA20, A34, Y36- amide Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACYGG-NH₂Acetyl-OSK1-K 1251 16, D20, A34, Y36,  G37, G38-amideGVIINVKCKISRQCLKPCK[Aad]AGMRFGKCMNGKCACYGC-NH₂ OSK1-K 16, 1252Aad20, A34, Y36, G37 G38-amideGVIINVKCKISRQCLKPCK[Aad]AGMRFGKCMNGKCHCYGG-NH₂ OSK1-K 16, 1253Aad20, Y36, G37, G38- amide GVIINVKCKISRQCLKPCK[Aad]AGMRFGKCMNGKCACYGGOSK1-K 16, 1254 Aad20, A34, Y36, G37, G38GVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCACYGG-NH₂ OSK1-H 125516, D20, A34, Y36,  G37, G38-amideGVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCACYGG OSK1-H 1256 16, D20, A34, Y36, G37, G38 GVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCACY-NH₂ Δ37-38-OSK1-H 125716, D20, A34, Y36- amide GVIINVKCKISRQCLHPCKDACMRFGKCMNGKCHCYGG-NH₂OSK1-H 1258 16, D20, A34, Y36,  G37, G38-amideGVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCHCYGG OSK1-H 1259 16, D20, A34, Y36, G37, G38 GVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCHCYPK OSK1-H 126016, D20, A34, Y36, GVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCAC Δ36-38 OSK1-H1261 16, D20, A34, Y36, GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC[1Nal]GG-OSK1-K 1262 NH₂ 16, D20, A34, 1Nal36, G37, G38-amideGVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC[1Nal]PK- OSK1-K 1263 NH₂16, D20, A34, 1Nal36- amide GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC[2Nal]GG-OSK1-K 1264 NH₂ 16, D20, A34, 2Nal36, G37, G38-amideGVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC[Cha]GG-NH₂ OSK1-K 126516, D20, A34, Cha36, G37, G38-amideGVIINVKCKISRQCLKPCKDAGMRFGKCMNCKCAC[MePhe]GG- OSK1-K 1266 NH₂16, D20, A34, MePhe36, G37, G38- amideGVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC[BiphA]GG- OSK1-K 1267 NH₂16, D20, A34, BiPhA36, G37, G38- amideGVIINVKCKISRQCLKPCKDAGMRFGKCMNGKC[Aib]CYGG-NH₂ OSK1-K 16, D20, 1268Aib34, Y36, G37,  G38-amideGVIINVKCKISRQCLKPCKDAGMRFGKCMNGKC[Abu]CYGG-NH₂ OSK1-K 16, D20, 1269Abu34, Y36, G37,  G38-amide GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC[1Nal]Δ37-38 OSK1-H 1270 16, D20, A34, 1Nal36,- amideGVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCAC[1Nal]GG- OSK1-H 1271 NH₂16, D20, A34, 1Nal36, G37, G38- amideGVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC[4Bip]-NH₂ Δ37-38 OSK1-H 127216, D20, A34, 4Bip 36, -amideGVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCAC[4Bip]GG- OSK1-H 1273 NH₂16, D20, A34, 4Bip 36, G37, G38- amideGVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCGGG OSK1-K16, E20, G36- 1274 38

Any of the sequences set forth in Table 7, can also be derivatized ateither its N-terminal or C-terminal with a fatty acid having from 4 to10 carbon atoms and from 0 to 2 carbon-carbon double bonds, or aderivative thereof such as an ω-amino-fatty acid. (E.g., Mouhat et al.,WO 2006/002850 A2, which is incorporated by reference in its entirety).Examples of such fatty acids include valeric acid or (for theC-terminal) ω-amino-valeric acid

TABLE 8 Pi2 peptide and PiP2 s peptide analog equences SEQ Short-hand IDSequence/structure designation NO: TISCTNPKQCYPHCKKETGYPNAKCMNRKCKCFGRPi2 17 TISCTNPXQCYPHCKKETGYPNAKCMNRKCKCFGR Pi2-X8 299TISCTNPAQCYPHCKKETGYPNAKCMNRKCKCFGR Pi2-A8 300TISCTNPKQCYPHCXKETGYPNAKCMNRKCKCFGR Pi2-X15 301TISCTNPKQCYPHCAKETGYPNAKCMNRKCKCFGR Pi2-A15 302TISCTNPKQCYPHCKXETGYPNAKCMNRKCKCFGR Pi2-X16 303TISCTNPKQCYPHCKAETGYPNAKCMNRKCKCFGR Pi2-A16 304TISCTNPKQCYPHCKKETGYPNAXCMNRKCKCFGR Pi2-X24 305TISCTNPKQCYPHCKKETGYPNAACMNRKCKCFGR Pi2-A24 306TISCTNPKQCYPHCKKETGYPNAKCMNXKCKCFGR Pi2-X28 307TISCTNPKQCYPHCKKETGYPNAKCMNAKCKCFGR Pi2-A28 308TISCTNPKQCYPHCKKETGYPNAKCMNRXCKCFGR Pi2-X29 309TISCTNPKQCYPHCKKETGYPNAKCMNRACKCFGR Pi2-A29 310TISCTNPKQCYPHCKKETGYPNAKCMNRKCXCFGR Pi2-X31 311TISCTNPKQCYPHCKKETGYPNAKCMNRKCACFGR Pi2-A31 312TISCTNPKQCYPHCKKETGYPNAKCMNRKCKCFGX Pi2-X35 313TISCTNPKQCYPHCKKETGYPNAKCMNRKCKCFGA Pi2-A35 314TISCTNPKQCYPHCKKETGYPNAKCMNRKCKCFG Pi2-d35 315

TABLE 9 Anuroctoxin (AnTx) peptide and peptide analog sequences SEQShort-hand ID Sequence/structure designation NO:ZKECTGPQHCTNFCRKNKCTHGKCMNRKCKCFNCK Anuroctoxin 62 (AnTx) KECTGPQHCTNFCRKNKCTHGKCMNRKCKCFNCK AnTx-d1 316 XECTGPQHCTNFCRKNKCTHGKCMNRKCKCFNCK AnTx-d1, X2 317 AECTGPQHCTNFCRKNKCTHGKCMNRKCKCFNCK AnTx-d1, A2 318

TABLE 10 Noxiustoxin (NTX) peptide and NTX peptide analog sequencesShort- hand SEQ desig- ID Sequence/structure nation NO:TIINVKCTSPKQCSKPCKELYGSSAGAKCMNGKCKCYNN NTX 30TIINVACTSPKQCSKPCKELYGSSAGAKCMNGKCKCYNN NTX-A6 319TIINVKCTSPKQCSKPCKELYGSSRGAKCMNGKCKCYNN NTX-R25 320TIINVKCTSSKQCSKPCKELYGSSAGAKCMNGKCKCYNN NTX-S10 321TIINVKCTSPKQCWKPCKELYGSSAGAKCMNGKCKCYNN NTX-W14 322TIINVKCTSPKQCSKPCKELYGSSGAXCMNGKCKCYNN NTX-A25d 323TIINVKCTSPKQCSKPCKELFGVDRGKCMNGKCKCYNN NTX- 324 IbTx1TIINVKCTSPKQCWKPCKELFGVDRGKCMNGKCKCYN NTX- 325 IBTX2

TABLE 11 Kaliotoxin1 (KTX1) peptide and KTX1 peptide analog sequencesShort- hand SEQ desig- ID Sequence/structure nation NO:GVEINVKCSGSPQCLKPCKDAGMRFGKCMNRKCHCTPK KTX1 24 VRIPVSCKHSGQCLKPCKDAGMRFGKCMNGKCDCTPK KTX2 326GVEINVSCSGSPQCLKPCKDAGMRFGKCMNRKCHCTPK KTX1-S7 327GVEINVACSGSPQCLKPCKDAGMRFGKCMNRKCHCTPK KTX1-A7 328

TABLE 12 IKCa1 inhibitor peptide sequences Short- hand SEQ desig- IDSequence/structure nation NO: VSCTGSKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX 20  QFTNVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS ChTx 36  QFTQESCTASNQCWSICKRLHNTNRGKCMNKKCRCYS ChTx-Lq2 329

TABLE 13 Maurotoxin (MTx) peptide amd MTx peptide analog sequencesShort-hand SEQ ID Sequence/structure designation NO:VSCTGSKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX 20VSCAGSKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX-A4 330VSCTGAKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX-A6 331VSCTGSADCYAPCRKQTGCPNAKCINKSCKCYGC MTX-A7 332VSCTGSKDCAAPCRKQTGCPNAKCINKSCKCYGC MTX-A10 333VSCTGSKDCYAPCQKQTGCPNAKCINKSCKCYGC MTX-Q14 334VSCTGSKDCYAPCRQQTGCPNAKCINKSCKCYGC MTX-Q15 335VSCTGSKDCYAPCQQQTGCPNAKCINKSCKCYGC MTX-Q14, 15 336VSCTGSKDCYAPCRKQTGCPNAKCINKSCKCYAC MTX-A33 337VSCTGSKDCYAPCRKQTGCPYGKCMNRKCKCNRC MTX-HsTx1 338VSCTGSKDCYAACRKQTGCANAKCINKSCKCYGC MTX-A12, 20 339 VSCTGSKDCYAPCRKQTGX^(M19)PNAKCINKSCKCYGX^(M34) MTX-X19, 34 340VSCTGSKDCYAPCRKQTGSPNAKCINKSCKCYGS MTX-S19, 34 341VSCTGSADCYAPCRKQTGCPNAKCINKSCKCYGC MTX-A7 342VVIGQRCTGSKDCYAPCRKQTGCPNAKCINKSCKCYGC TsK-MTX 343VSCRGSKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX-R4 1301VSCGGSKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX-G4 1302VSCTTSKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX-T5 1304VSCTASKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX-A5 1305VSCTGTKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX-T6 1306VSCTGPKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX-P6 1307VSCTGSKDCGAPCRKQTGCPNAKCINKSCKCYGC MTX-G10 1309VSCTGSKDCYRPCRKQTGCPNAKCINKSCKCYGC MTX-R11 1311VSCTGSKDCYDPCRKQTGCPNAKCINKSCKCYGC MTX-D11 1312VSCTGSKDCYAPCRKRTGCPNAKCINKSCKCYGC MTX-R16 1315VSCTGSKDCYAPCRKETGCPNAKCINKSCKCYGC MTX-E16 1316VSCTGSKDCYAPCRKQTGCPYAKCINKSCKCYGC MTX-Y21 1317VSCTGSKDCYAPCRKQTGCPNSKCINKSCKCYGC MTX-S22 1318VSCTGSKDCYAPCRKQTGCPNGKCINKSCKCYGC MTX-G22 1319VSCTGSKDCYAPCRKQTGCPNAKCINRSCKCYGC MTX-R27 1320VSCTGSKDCYAPCRKQTGCPNAKCINKTCKCYGC MTX-T28 1321VSCTGSKDCYAPCRKQTGCPNAKCINKMCKCYGC MTX-M28 1322VSCTGSKDCYAPCRKQTGCPNAKCINKKCKCYGC MTX-K28 1323VSCTGSKDCYAPCRKQTGCPNAKCINKSCKCNGC MTX-N32 1324VSCTGSKDCYAPCRKQTGCPNAKCINKSCKCYRC MTX-R33 1325VSCTGSKDCYAPCRKQTGCPNAKCINKSCKCYGCS MTX-S35 1326 SCTGSKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX-d1 1327 SCTGSKDCYAPCRKQTGCPNAKCINKSCKCYGCS MTX-S35 d1 1328VSCTGSKDCYAPCAKQTGCPNAKCINKSCKCYGC MTX-A14 1329VSCTGSKDCYAPCRAQTGCPNAKCINKSCKCYGC MTX-A15 1330VSCTGSKDCYAPCRKQTGCPNAACINKSCKCYGC MTX-A23 1331VSCTGSKDCYAPCRKQTGCPNAKCINASCKCYGC MTX-A27 1332VSCTGSKDCYAPCRKQTGCPNAKCINKSCACYGC MTX-A30 1333VSCTGSKDCYAPCRKQTGCPNAKCINKSCKCAGC MTX-A32 1334ASCTGSKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX-A1 1335MSCTGSKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX-M1 1336

In Table 13 and throughout this specification, X^(m19) and X^(m34) areeach independently nonfunctional residues.

TABLE 14 Charybdotoxin(ChTx) peptide and ChTx peptide analog sequencesShort- hand SEQ desig- ID Sequence/structure nation NO:QFTNVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS ChTx 36QFTNVSCTTSKECWSVCQRLHNTSRGKCMNKECRCYS ChTx- 59 E32QFTNVSCTTSKECWSVCQRLHNTSRGKCMNKDCRCYS ChTx- 344 D32      CTTSKECWSVCQRLHNTSRGKCMNKKCRCYS ChTx- 345 d1-d6QFTNVSCTTSKECWSVCQRLFGVDRGKCMGKKCRCYQ ChTx- 346 IbTxQFTNVSCTTSKECWSVCQRLHNTSRGKCMNGKCRCYS ChTx- 1369 G31QFTNVSCTTSKECLSVCQRLHNTSRGKCMNKKCRCYS ChTx- 1370 L14QFTNVSCTTSKECASVCQRLHNTSRGKCMNKKCRCYS ChTx- 1371 A14QFTNVSCTTSKECWAVCQRLHNTSRGKCMNKKCRCYS ChTx- 1372 A15QFTNVSCTTSKECWPVCQRLHNTSRGKCMNKKCRCYS ChTx- 1373 P15QFTNVSCTTSKECWSACQRLHNTSRGKCMNKKCRCYS ChTx- 1374 A16QFTNVSCTTSKECWSPCQRLHNTSRGKCMNKKCRCYS ChTx- 1375 P16QFTNVSCTTSKECWSVCQRLHNTSAGKCMNKKCRCYS ChTx- 1376 A25QFTNVACTTSKECWSVCQRLHNTSRGKCMNKKCRCYS ChTx- 1377 A6QFTNVKCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS ChTx- 1378 K6QFTNVSCTTAKECWSVCQRLHNTSRGKCMNKKCRCYS ChTx- 1379 A10QFTNVSCTTPKECWSVCQRLHNTSRGKCMNKKCRCYS ChTx- 1380 P10QFTNVSCTTSKACWSVCQRLHNTSRGKCMNKKCRCYS ChTx- 1381 A12QFTNVSCTTSKQCWSVCQRLHNTSRGKCMNKKCRCYS ChTx- 1382 Q12AFTNVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS ChTx- 1383 A1TFTNVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS ChTx- 1384 T1QATNVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS ChTx- 1385 A2QITNVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS ChTx- 1386 I2QFANVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS ChTx- 1387 A3QFINVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS ChTx- 1388 I3TIINVKCTSPKQCLPPCKAQFGTSRGKCMNKKCRCYSP ChTx- 1389 MgTxTIINVSCTSPKQCLPPCKAQFGTSRGKCMNKKCRCYSP ChTx- 1390 MgTx-b

TABLE 15 SKCa inhibitor peptide sequences SEQ Short-hand IDSequence/structure designation NO:            CNCKAPETALCARRCQQHG Apamin68 AFCNLRMCQLSCRSLGLLGKCIGDKCECVKH ScyTx 51    AVCNLKRCQLSCRSLGLLGKCIGDKCECVKHG BmP05 50     TVCNLRRCQLSCRSLGLLGKCIGVKCECVKH P05 52     AFCNLRRCELSCRSLGLLGKCIGEECKCVPY Tamapin 53VSCEDCPEHCSTQKAQAKCDNDKCVCEPI P01 16 VVIGQRCYRSPDCYSACKKLVGKATGKCTNGRCDCTsK 47

TABLE 16 Apamin peptide and peptide analog inhibitor sequencesShort-hand SEQ ID Sequence/structure designation NO:           CNCKAPETALCARRCQQHG Apamin (Ap) 68 CNCXAPETALCARRCQQHG Ap-X4348 CNCAAPETALCARRCQQHG Ap-A4 349 CNCKAPETALCAXRCQQHG Ap-X13 350CNCKAPETALCAARCQQHG Ap-A13 351 CNCKAPETALCARXCQQHG Ap-X14 352CNCKAPETALCARACQQHG Ap-A14 353

TABLE 17 Scyllatoxin (ScyTx), BmP05, P05, Tamapin, P01peptide and peptide analog inhibitor sequences SEQ Short-hand IDSequence/structure designation NO: AFCNLRMCQLSCRSLGLLGKCIGDKCECVKH ScyTx51 AFCNLRRCQLSCRSLGLLGKCIGDKCECVKH ScyTx-R7 354AFCNLRMCQLSCRSLGLLGKCMGKKCRCVKH ScyTx-IbTx 355AFSNLRMCQLSCRSLGLLGKSIGDKCECVKH ScyTx-C/S 356     AFCNLRRCELSCRSLGLLGKCIGEECKCVPY Tamapin 53

TABLE 18 BKCa inhibitor peptide sequences Short- hand SEQ desig- IDSequence/structure nation NO: QFTDVDCSVSKECWSVCKDLFGVDRGKCMGKKCRCYQ IbTx38   TFIDVDCTVSKECWAPCKAAFGVDRGKCMGKKCKCYV Slo- 39 toxin (Slo- TX)QFTDVKCTGSKQCWPVCKQMFGKPNGKCMNGKCRCYS BmTx1 40WCSTCLDLACGASRECYDPCFKAFGRAHGKCMNNKCRCYTN BuTx 41  FGLIDVKCFASSECWTACKKVTGSGQGKCQNNQCRCY Marten- 35 Tx  ITINVKCTSPQQCLRPCKDRFGQHAGGKCINGKCKCYP CIITx1 29

TABLE 19 IbTx, Slotoxin, BmTx1, & BuTX (Slotoxin family)peptide and peptide analog inhibitor sequences Short- hand SEQ desig- IDSequence/structure nation NO: QFTDVDCSVSKECWSVCKDLFGVDRGKCMGKKCRCYQ IbTx38 QFTDVDCSVSXECWSVCKDLFGVDRGKCMGKKCRCYQ IbTx- 357 X11QFTDVDCSVSAECWSVCKDLFGVDRGKCMGKKCRCYQ IbTx- 358 A11QFTDVDCSVSKECWSVCXDLFGVDRGKCMGKKCRCYQ IbTx- 359 X18QFTDVDCSVSKECWSVCADLFGVDRGKCMGKKCRCYQ IbTx- 360 A18QFTDVDCSVSKECWSVCKDLFGVDXGKCMGKKCRCYQ IbTx- 361 X25QFTDVDCSVSKECWSVCKDLFGVDAGKCMGKKCRCYQ IbTx- 362 A25QFTDVDCSVSKECWSVCKDLFGVDRGXCMGKKCRCYQ IbTx- 363 X27QFTDVDCSVSKECWSVCKDLFGVDRGACMGKKCRCYQ IbTx- 364 A27QFTDVDCSVSKECWSVCKDLFGVDRGKCMGXKCRCYQ IbTx- 365 X31QFTDVDCSVSKECWSVCKDLFGVDRGKCMGAKCRCYQ IbTx- 366 A31QFTDVDCSVSKECWSVCKDLFGVDRGKCMGKXCRCYQ IbTx- 367 X32QFTDVDCSVSKECWSVCKDLFGVDRGKCMGKACRCYQ IbTx- 368 A32QFTDVDCSVSKECWSVCKDLFGVDRGKCMGKKCXCYQ IbTx- 369 X34QFTDVDCSVSKECWSVCKDLFGVDRGKCMGKKCACYQ IbTx- 370 A34QFTDVKCTGSKQCWPVCKQMFGKPNGKCMNGKCRCYS BmTx1 371QFTDVXCTGSKQCWPVCKQMFGKPNGKCMNGKCRCYS BmTx1- 372 X6QFTDVACTGSKQCWPVCKQMFGKPNGKCMNGKCRCYS BmTx1- 373 A6QFTDVKCTGSXQCWPVCKQMFGKPNGKCMNGKCRCYS BmTx1- 374 X11QFTDVKCTGSAQCWPVCKQMFGKPNGKCMNGKCRCYS BmTx1- 375 A11QFTDVKCTGSKQCWPVCXQMFGKPNGKCMNGKCRCYS BmTx1- 376 X18QFTDVKCTGSKQCWPVCAQMFGKPNGKCMNGKCRCYS BmTx1- 377 A18QFTDVKCTGSKQCWPVCKQMFGXPNGKCMNGKCRCYS BmTx1- 378 X23QFTDVKCTGSKQCWPVCKQMFGAPNGKCMNGKCRCYS BmTx1- 379 A23QFTDVKCTGSKQCWPVCKQMFGKPNGXCMNGKCRCYS BmTx1- 380 X27QFTDVKCTGSKQCWPVCKQMFGKPNGACMNGKCRCYS BmTx1- 381 A27QFTDVKCTGSKQCWPVCKQMFGKPNGKCMNGXCRCYS BmTx1- 382 X32QFTDVKCTGSKQCWPVCKQMFGKPNGKCMNGARCYS BmTx1- 383 A32QFTDVKCTGSKQCWPVCKQMFGKPNGKCMNGKCXYS BmTx1- 384 X34QFTDVKCTGSKQCWPVCKQMFGKPNGKCMNGKCAYS BmTx1- 385 A34WCSTCLDLACGASRECYDPCFKAFGRAHGKCMNNKCRCYTN BuTx 386WCSTCLDLACGASXCYDPCFKAFGRAHGKCMNNKCRCYTN BuTx- 387 X14WCSTCLDLACGASACYDPCFKAFGRAHGKCMNNKCRCYTN BuTx- 388 A14WCSTCLDLACGASRECYDPCFXFGRAHGKCMNNKCRCYTN BuTx- 389 X22WCSTCLDLACGASRECYDPCFAGRAHGKCMNNKCRCYTN BuTx- 390 A22WCSTCLDLACGASRECYDPCFKAFGXHGKCMNNKCRCYTN BuTx- 391 X26WCSTCLDLACGASRECYDPCFKAFGAHGKCMNNKCRCYTN BuTx- 392 A26WCSTCLDLACGASRECYDPCFKAFGRAHGXMNNKCRCYTN BuTx- 393 X30WCSTCLDLACGASRECYDPCFKAFGRAHGAMNNKCRCYTN BuTx- 394 A30WCSTCLDLACGASRECYDPCFKAFGRAHGKCMNNXRCYTN BuTx- 395 X35WCSTCLDLACGASRECYDPCFKAFGRAHGKCMNNARCYTN BuTx- 396 A35WCSTCLDLACGASRECYDPCFKAFGRAHGKCMNNKCXYTN BuTx- 397 X37WCSTCLDLACGASRECYDPCFKAFGRAHGKCMNNKCAYTN BuTx- 398 A37

TABLE 20 Martentoxin peptide and peptide analog inhibitor sequencesShort- hand SEQ desig- ID Sequence/structure nation NO:FGLIDVKCFASSECWTACKKVTGSGQGKCQNNQCRCY MartenTx 35FGLIDVXCFASSECWTACKKVTCSGQGKCQNNQCRCY MartenTx- 399 X7FGLIDVACFASSECWTACKKVTGSGQGKCQNNQCRCY MartenTx- 400 A7FGLIDVKCFASSECWTACXKVTGSGQGKCQNNQCRCY MartenTx- 401 X19FGLIDVKCFASSECWTACAKVTGSGQGKCQNNQCRCY MartenTx- 402 A19FGLIDVKCFASSECWTACKXVTGSGQGKCQNNQCRCY MartenTx- 403 X20FGLIDVKCFASSECWTACKAVTGSGQGKCQNNQCRCY MartenTx- 404 A20FGLIDVKCFASSECWTACKKVTGSGQGXCQNNQCRCY MartenTx- 405 X28FGLIDVKCFASSECWTACKKVTGSGQGACQNNQCRCY MartenTx- 406 A28FGLIDVKCFASSECWTACKKVTGSGQGKCQNNQCXCY MartenTx- 407 X35FGLIDVKCFASSECWTACKKVTGSGQGKCQNNQCACY MartenTx- 408 A35

TABLE 21 N type Ca²⁺ channel inhibitor peptide sequences SEQ Short-handID Sequence/structure designation NO: CKGKGAKCSRLMYDCCTGSCRSGKC MVIIA 65CKSPGSSCSPTSYNCCRSCNPYTKRCY GVIA 64 CKSKGAKCSKLMYDCCTGSCSGTVGRC CVIA 409CKLKGQSCRKTSYDCCSGSCGRSGKC SVIB 347 AEKDCIAPGAPCFGTDKPCCNPRAWCSSYANKCLPtu1 66 CKGKGASCRKTMYDCCRGSCRSGRC CVIB 1364 CKGKGQSCSKLMYDCCTGSCSRRGKCCVIC 1365 CKSKGAKCSKLMYDCCSGSCSGTVGRC CVID 1366CLSXGSSCSXTSYNCCRSCNXYSRKCY TVIA 1367

TABLE 22 ωOMVIIA peptide and peptide analog inhibitor sequencesShort-hand SEQ ID Sequence/structure designation NO:CKGKGAKCSRLMYDCCTGSCRSGKC MVIIA 65 CXGKGAKCSRLMYDCCTGSCRSGKC MVIIA-X2410 CAGKGAKCSRLMYDCCTGSCRSGKC MVIIA-A2 411 CKGXGAKCSRLMYDCCTGSCRSGKCMVIIA-X4 412 CKGAGAKCSRLMYDCCTGSCRSGKC MVIIA-A4 413CKGKGAXCSRLMYDCCTGSCRSGKC MVIIA-X7 414 CKGKGAACSRLMYDCCTGSCRSGKCMVIIA-A7 415 CKGKGAKCSXLMYDCCTGSCRSGKC MVIIA-X10 416CKGKGAKCSALMYDCCTGSCRSGKC MVIIA-A10 417 CKGKGAKCSRLMYDCCTGSCXSGKCMVIIA-X21 418 CKGKGAKCSRLMYDCCTGSCASGKC MVIIA-A21 419CKGKGAKCSRLMYDCCTGSCRSGXC MVIIA-X24 420 CKGKGAKCSRLMYDCCTGSCRSGACMVIIA-A24 421

TABLE 23 ωGVIA peptide and peptide analog inhibitor sequences Short-handSEQ ID Sequence/structure designation NO: CKSPGSSCSPTSYNCCRSCNPYTKRCYGVIA 64 CXSPGSSCSPTSYNCCRSCNPYTKRCY GVIA-X2 422CASPGSSCSPTSYNCCRSCNPYTKRCY GVIA-A2 423 CKSPGSSCSPTSYNCCXSCNPYTKRCYGVIA-X17 424 CKSPGSSCSPTSYNCCASCNPYTKRCY GVIA-A17 425CKSPGSSCSPTSYNCCRSCNPYTXRCY GVIA-X24 426 CKSPGSSCSPTSYNCCRSCNPYTARCYGVIA-A24 427 CKSPGSSCSPTSYNCCRSCNPYTKXCY GVIA-X25 428CKSPGSSCSPTSYNCCRSCNPYTKACY GVIA-A25 429

TABLE 24 Ptu1 peptide and peptide analog inhibitor sequences SEQShort-hand ID Sequence/structure designation NO:AEKDCIAPGAPCFGTDKPCCNPRAWCSSYANKCL Ptu1 66AEXDCIAPGAPCFGTDKPCCNPRAWCSSYANKCL Ptu1-X3 430AEADCIAPGAPCFGTDKPCCNPRAWCSSYANKCL Ptu1-A3 431AEKDCIAPCAPCFGTDXPCCNPRAWCSSYANKCL Ptu1-X17 432AEKDCIAPGAPCFGTDAPCCNPRAWCSSYANKCL Ptu1-A17 433AEKDCIAPGAPCFGTDKPCCNPXAWCSSYANKCL Ptu1-X23 434AEKDCIAPGAPCFGTDKPCCNPAAWCSSYANKCL Ptu1-A23 435AEKDCIAPGAPCFGTDKPCCNPRAWCSSYANXCL Ptu1-X32 436AEKDCIAPGAPCFGTDKPCCNPRAWCSSYANACL Ptu1-A32 437

TABLE 25 Thrixopelma pruriens (ProTx1) and ProTx1 pep-tide analogs and other T type Ca²⁺ channel in- hibitor peptide sequencesShort- hand SEQ desig- ID Sequence/structure nation NO:ECRYWLGGCSAGQTCCKHLVCSRRHGWCVWDGTFS ProTx1 56ECXYWLGGCSAGQTCCKHLVCSRRHGWCVWDGTFS ProTx1-X3 438ECAYWLGGCSAGQTCCKHLVCSRRHGWCVWDGTFS ProTx1-A3 439ECRYWLGGCSAGQTCCXHLVCSRRHGWCVWDGTFS ProTx1- 440 X17ECRYWLGGCSAGQTCCAHLVCSRRHGWCVWDGTFS ProTx1- 441 A17ECRYWLGGCSAGQTCCKHLVCSXRHGWCVWDGTFS ProTx1- 442 X23ECRYWLGGCSAGQTCCKHLVCSARHGWCVWDGTFS ProTx1- 443 A23ECRYWLGGCSAGQTCCKHLVCSRXHGWCVWDGTFS ProTx1- 444 X24ECRYWLGGCSAGQTCCKHLVCSRAHGWCVWDGTFS ProTx1- 445 A24KIDGYPVDYW NCKRICWYNN KYCNDLCKGL Kurtoxin 1276KADSGYCWGW TLSCYCQGLP DNARIKRSGR CRA

TABLE 26 BeKM1 M current inhibitor peptide and BeKM1peptide analog sequences SEQ Short-hand ID Sequence/structuredesignation NO: RPTDIKCSESYQCFPVCKSRFGKTNGRCVNGFCDCF BeKM1 63 PTDIKCSESYQCFPVCKSRFGKTNGRCVNGFCDCF BeKM1-d1 446XPTDIKCSESYQCFPVCKSRFGKTNGRCVNGFCDCF BeKM1-X1 447APTDIKCSESYQCFPVCKSRFGKTNGRCVNGFCDCF BeKM1-A1 448RPTDIXCSESYQCFPVCKSRFGKTNGRCVNGFCDCF BeKM1-X6 449RPTDIACSESYQCFPVCKSRFGKTNGRCVNGFCDCF BeKM1-A6 450RPTDIKCSESYQCFPVCXSRFGKTNCRCVNGFCDCF BeKM1-X18 451RPTDIKCSESYQCFPVCASRFGKTNGRCVNGFCDCF BeKM1-A18 452RPTDIKCSESYQCFPVCKSXFGKTNGRCVNGFCDCF BeKM1-X20 453RPTDIKCSESYQCFPVCKSAFGKTNGRCVNGFCDCF BeKM1-A20 454RPTDIKCSESYQCFPVCKSRFGXTNGRCVNGFCDCF BeKM1-X23 455RPTDIKCSESYQCFPVCKSRFGATNGRCVNGFCDCF BeKM1-A23 456RPTDIKCSESYQCFPVCKSRFGKTNGXCVNGFCDCF BeKM1-X27 457RPTDIKCSESYQCFPVCKSRFGKTNGACVNGFCDCF BeKM1-A27 458

TABLE 27 Na⁺ channel inhibitor peptide sequences Short-hand SEQ IDSequence/structure designation NO: QRCCNGRRGCSSRWCRDHSRCC SmIIIa 459RDCCTOOKKCKDRQCKOQRCCA μ-GIIIA 460 RDCCTOORKCKDRRCKOMRCCA μ-GIIIB 461ZRLCCGFOKSCRSRQCKOHRCC μ-PIIIA 462 ZRCCNGRRGCSSRWCRDHSRCC μ-SmIIIA 463ACRKKWEYCIVPIIGFIYCCPGLICGPFVCV μO-MrVIA 464ACSKKWEYCIVPIIGFIYCCPGLICGPFVCV μO-MrVIB 465EACYAOGTFCGIKOGLCCSEFCLPGVCFG δ-PVIA 466 DGCSSGGTFCGIHOGLCCSEFCFLWCITFIDδ-SVIE 467 WCKQSGEMCNLLDQNCGDGYCIVLVCT δ-TxVIA 468VKPCRKEGQLCDPIFQNCCRGWNCVLFCV δ-GmVIA 469

TABLE 28 Cl- channel inhibitor peptide sequences Short- hand SEQ desig-ID Sequence/structure nation NO:   MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLCRCTX 67 MCMPCFTTDHQMAXKCDDCCGGKGRGKCYGPQCLCR CTX- 470 X14MCMPCFTTDHQMAAKCDDCCGGKGRGKCYGPQCLCR CTX- 471 A14MCMPCFTTDHQMARXCDDCCGGKGRGKCYGPQCLCR CTX- 472 X15MCMPCFTTDHQMARACDDCCGGKGRGKCYGPQCLCR CTX- 473 A15MCMPCFTTDHQMARKCDDCCGGXGRGKCYGPQCLCR CTX- 474 X23MCMPCFTTDHQMARKCDDCCGGAGRGKCYGPQCLCR CTX- 475 A23MCMPCFTTDHQMARKCDDCCGGKGXGKCYGPQCLCR CTX- 476 X25MCMPCFTTDHQMARKCDDCCGGKGAGKCYGPQCLCR CTX- 477 A25MCMPCFTTDHQMARKCDDCCGGKGRGXCYGPQCLCR CTX- 478 X27MCMPCFTTDHQMARKCDDCCGGKGRGACYGPQCLCR CTX- 479 A27  MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLCX CTX- 480 X36MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLCA CTX- 481 A36   MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLC CTX- 482 d36QTDGCGPCFTTDANMARKCRECCGGNGKCFGPQCLCNRE Bm-12b 483QTDGCGPCFTTDANMAXKCRECCGGNGKCFGPQCLCNRE Bm- 484 12b- X17QTDGCGPCFTTDANMAAKCRECCGGNGKCFGPQCLCNRE Bm- 485 12b- A17QTDGCGPCFTTDANMARXCRECCGGNGKCFGPQCLCNRE Bm- 486 12b- X18QTDGCGPCFTTDANMARACRECCGGNGKCFGPQCLCNRE Bm- 487 12b- A18QTDGCGPCFTTDANMARKCXECCGGNGKCFGPQCLCNRE Bm- 488 12b- X20 QTDGCGPCFTTDANMARKCAECCGGNGKCFGPQCLCNRE Bm- 489 12b- A20QTDGCGPCFTTDANMARKCRECCGGNGXCFGPQCLCNRE Bm- 490 12b- X28QTDGCGPCFTTDANMARKCRECCGGNGACFGPQCLCNRE Bm- 491 12b- A28QTDGCGPCFTTDANMARKCRECCGGNGKCFGPQCLCNXE Bm- 492 12b- X38QTDGCGPCFTTDANMARKCRECCGGNGKCFGPQCLCNAE Bm- 493 12b- A38

TABLE 29 Kv2.1 inhibitor peptide sequences SEQ Short-hand IDSequence/structure designation NO: ECRYLFGGCKTTSDCCKHLGCKFRDKYCAWDFTFSHaTx1 494 ECXYLFGGCKTTSDCCKHLGCKFRDKYCAWDFTFS HaTx1-X3 495ECAYLFGGCKTTSDCCKHLGCKFRDKYCAWDFTFS HaTx1-A3 496ECRYLFGGCXTTSDCCKHLGCKFRDKYCAWDFTFS HaTx1-X10 497ECRYLFGGCATTSDCCKHLGCKFRDKYCAWDFTFS HaTx1-A10 498ECRYLFGGCKTTSDCCXHLGCKFRDKYCAWDFTFS HaTx1-X17 499ECRYLFGGCKTTSDCCAHLGCKFRDKYCAWDFTFS HaTx1-A17 500ECRYLFGGCKTTSDCCKHLGCXFRDKYCAWDFTFS HaTx1-X22 501ECRYLFGGCKTTSDCCKHLGCAFRDKYCAWDFTFS HaTx1-A22 502ECRYLFGGCKTTSDCCKHLGCKFXDKYCAWDFTFS HaTx1-X24 503ECRYLFGGCKTTSDCCKHLGCKFADKYCAWDFTFS HaTx1-A24 504ECRYLFGGCKTTSDCCKHLGCKFRDXYCAWDFTFS HaTx1-X26 505ECRYLFGGCKTTSDCCKHLGCKFRDAYCAWDFTFS HaTx1-A26 506

TABLE 30 Kv4.3 & Kv4.2 inhibitor peptide sequences Short-hand SEQ IDSequence/structure designation NO: YCQKWMWTCDEERKCCEGLVCRLWCKRIINM PaTx257 YCQXWMWTCDEERKCCEGLVCRLWCKRIINM PaTx2-X4 507YCQAWMWTCDEERKCCEGLVCRLWCKRIINM PaTx2-A4 508YCQKWMWTCDEEXKCCEGLVCRLWCKRIINM PaTx2-X13 509YCQKWMWTCDEEAKCCEGLVCRLWCKRIINM PaTx2-A13 510YCQKWMWTCDEERXCCEGLVCRLWCKRIINM PaTx2-X14 511YCQKWMWTCDEERACCEGLVCRLWCKRIINM PaTx2-A14 512YCQKWMWTCDEERKCCEGLVCXLWCKRIINM PaTx2-X22 513YCQKWMWTCDEERKCCEGLVCALWCKRIINM PaTx2-A22 514YCQKWMWTCDEERKCCEGLVCRLWCXRIINM PaTx2-X26 515YCQKWMWTCDEERKCCEGLVCRLWCARIINM PaTx2-A26 516YCQKWMWTCDEERKCCEGLVCRLWCKXIINM PaTx2-X27 517YCQKWMWTCDEERKCCEGLVCRLWCKAIINM PaTx2-A27 518

TABLE 31 nACHR channel inhibitor peptide sequences SEQ Short-hand IDSequence/structure designation NO: GCCSLPPCAANNPDYC PnlA 519GCCSLPPCALNNPDYC PnlA-L10 520 GCCSLPPCAASNPDYC PnlA-S11 521GCCSLPPCALSNPDYC PnlB 522 GCCSLPPCAASNPDYC PnlB-A10 523 GCCSLPPCALNNPDYCPnlB-N11 524 GCCSNPVCHLEHSNLC Mll 525 GRCCHPACGKNYSC α-Ml 526RD(hydroxypro)CCYHPTCNMSNPQIC α-El 527 GCCSYPPCFATNPDC α-AulB 528RDPCCSNPVCTVHNPQIC α-PlA 529 GCCSDPRCAWRC α-lml 530 ACCSDRRCRWRC α-lmll531 ECCNPACGRHYSC α-Gl 532 GCCGSY(hydroxypro)NAACH αA-PIVA 533(hydroxypro)CSCKDR(hydroxypro) SYCGQ GCCPY(hydroxypro)NAACH  αA-EIVA 534(hydroxypro)CGCKVGR (hydroxypro)(hydroxypra)YCDR (hydroxypro)SGGH(hydroxypro)(hydroxypro) ψ-PIIIE 535 CCLYGKCRRY (hydroxypro)GCSSASCCQRGCCSDPRCNMNNPDYC EpI 536 GCCSHPACAGNNQHIC GIC 537IRD(γ-carboxyglu) CCSNPACRVNN GID 538 (hydroxypro)HVC GGCCSHPACAANNQDYCAnIB 539 GCCSYPPCFATNSDYC AuIA 540 GCCSYPPCFATNSGYC AuIC 541

TABLE 32 Agelenopsis aperta (Agatoxin) toxin peptides and peptideanalogs and other Ca²⁺ channel inhibiter peptides Short-hand SEQ IDSequence/structure designation NO: KKKCIAKDYG RCKWGGTPCC RGRGCICSIMω-Aga-IVA 959 GTNCECKPRL IMEGLGLA EDNCIAEDYG KCTWGGTKCC RGRPCRCSMIω-Aga-IVB 960 GTNCECTPRL IMEGLSFA SCIDIGGDCD GEKDDCQCCR RNGYCSCYSLω-Aga-IIIA 961 FGYLKSGCKC VVGTSAEFQG ICRRKARQCY NSDPDKCESH NKPKRRSCIDIGGDCD GEKDDCQCCR RNGYCSCYSL ω-Aga-IIIA- 962FGYLKSGCKC VVGTSAEFQG ICRRKARTCY T58 NSDPDKCESH NKPKRRSCIDFGGDCD GEKDDCQCCR SNGYCSCYSL ω-Aga-IIIB 963FGYLKSGCKC EVGTSAEFRR ICRRKAKQCY NSDPDKCVSV YKPKRRSCIDFGGDCD GEKDDCQCCR SNGYCSCYNL ω-Aga-IIIB- 964 FGYLKSGCKC EVGTSAEFRRN29 ICRRKAKQCYNSDPDKCVSV YKPKRR SCIDFGGDCD GEKDDCQCCR SNGYCSCYNLω-Aga-IIIB- 965 FGYLRSGCKC EVGTSAEFRR ICRRKAKQCY N29/R35NSDPDKCVSV YKPKRR NCIDFGGDCD GEKDDCQCCX RNGYCSCYNL ω-Aga-IIIC 966FGYLKRGCKX EVG SCIKIGEDCD GDKDDCQCCR TNGYCSXYXL FGYLKSG ω-Aga-IIID 967GCIEIGGDCD GYQEKSYCQC CRNNGFCS ω-Aga-IIA 968AKAL PPGSVCDGNE SDCKCYGKWH KCRCPWKWHF ω-Aga-IA 969TGEGPCTCEK GMKHTCITKL HCPNKAEWGL DW (major chain)ECVPENGHCR DWYDECCEGF YCSCRQPPKC ICRNNNX μ-Aga 970DCVGESQQCA DWAGPHCCDG YYCTCRYFPK CICVNNN μ-Aga-6 971ACVGENKQCA DWAGPHCCDG YYCTCRYFPK CICRNNN μ-Aga-5 972ACVGENQQCA DWAGPHCCDG YYCTCRYFPK CICRNNN μ-Aga-4 973ADCVGDGQRC ADWAGPYCCS GYYCSCRSMP μ-Aga-3 1275 YCRCRSDSECATKNKRCA DWAGPWCCDG LYCSCRSYPG CMCRPSS μ-Aga-2 974ECVPENGHCR DWYDECCEGF YCSCRQPPKC ICRNNN μ-Aga-1 975AELTSCFPVGHECDGDASNCNCCGDDVYCGCGWGRWNCKC Tx-1 1277KVADQSYAYGICKDKVNCPNRHLWPAKVCKKPCRREC GCANAYKSCNGPHTCCWGYNGYKKACICSGXNWKTx3-3 1278 SCINVGDFCDGKKDCCQCDRDNAFCSCSVIFGYKTNCRCE Tx3-4 1279SCINVGDFCDGKKDDCQCCRDNAFCSCSVIFGYKTNCRCE ω-PtXIIA 1280VGTTATSYGICMAKHKCGRQTTCTKPCLSKRCKKNHAECLMIGDTSCVPRLGRRCCYGAWCYCDQQLSCRRVGRKR Dw13.3 1281ECGWVEVNCKCGWSWSQRIDDWRADYSCKCPEDQ GGCLPHNRFCNALSGPRCCSGLKCKELSIWDSRCLAgelenin 1282 DCVRFWGKCSQTSDCCPHLACKSKWPRNICVWDGSV ω-GTx-SIA 1283GCLEVDYFCG IPFANNGLCC SGNCVFVCTP Q ω-conotoxin 1284 PnVIADDDCEPPGNF CGMIKIGPPC CSGWCFFACA ω-conotoxin 1285 PnVIB VCCGYKLCHP CLambda- 1286 conotoxin CMrVIA MRCLPVLIIL LLLTASAPGV VVLPKTEDDV Lambda-1287 PMSSVYGNGK SILRGILRNG VCCGYKLCHP C conotoxin CMrVIBKIDGYPVDYW NCKRICWYNN KYCNDLCKGL Kurtoxin 1276KADSGYCWGW TLSCYCQGLP DNARIKRSGR CRA CKGKGAPCRKTMYDCCSGSCGRRGKC MVIIC1368

In accordance with this invention are molecules in which at least one ofthe toxin peptide (P) portions of the molecule comprises a Kv1.3antagonist peptide. Amino acid sequences selected from ShK, HmK, MgTx,AgTx1, AgTx2, Heterometrus spinnifer (HsTx1), OSK1, Anuroctoxin (AnTx),Noxiustoxin (NTX), KTX1, Hongotoxin, ChTx, Titystoxin, BgK, BmKTX, BmTx,AeK, AsKS Tc30, Tc32, Pi1, Pi2, and/or Pi3 toxin peptides and peptideanalogs of any of these are preferred. Examples of useful Kv1.3antagonist peptide sequences include those having any amino acidsequence set forth in Table 1, Table 2, Table 3, Table 4, Table 5, Table6, Table 7, Table 8, Table 9, Table 10, and/or Table 11 herein above;

Other embodiments of the inventive composition include at least onetoxin peptide (P) that is an IKCa1 antagonist peptide. Useful IKCa1antagonist peptides include Maurotoxin (MTx), ChTx, peptides and peptideanalogs of either of these, examples of which include those having anyamino acid sequence set forth in Table 12, Table 13, and/or Table 14;

Other embodiments of the inventive composition include at least onetoxin peptide (P) that is a SKCa inhibitor peptide. Useful SKCainhibitor peptides include, Apamin, ScyTx, BmP05, P01, P05, Tamapin,TsK, and peptide analogs of any of these, examples of which includethose having any amino acid sequence set forth in Table 15;

Other embodiments of the inventive composition include at least onetoxin peptide (P) that is an apamin peptide, and peptide analogs ofapamin, examples of which include those having any amino acid sequenceset forth in Table 16;

Other embodiments of the inventive composition include at least onetoxin peptide (P) that is a Scyllotoxin family peptide, and peptideanalogs of any of these, examples of which include those having anyamino acid sequence set forth in Table 17;

Other embodiments of the inventive composition include at least onetoxin peptide (P) that is a BKCa inhibitor peptide, examples of whichinclude those having any amino acid sequence set forth in Table 18;

Other embodiments of the inventive composition include at least onetoxin peptide (P) that is a Slotoxin family peptide, and peptide analogsof any of these, examples of which include those having any amino acidsequence set forth in Table 19;

Other embodiments of the inventive composition include at least onetoxin peptide (P) that is a Martentoxin peptide, and peptide analogsthereof, examples of which include those having any amino acid sequenceset forth in Table 20;

Other embodiments of the inventive composition include at least onetoxin peptide (P) that is a N-type Ca²⁺ channel inhibitor peptide,examples of which include those having any amino acid sequence set forthin Table 21;

Other embodiments of the inventive composition include at least onetoxin peptide (P) that is a ωMVIIA peptide, and peptide analogs thereof,examples of which include those having any amino acid sequence set forthin Table 22;

Other embodiments of the inventive composition include at least onetoxin peptide (P) that is a ωGVIA peptide, and peptide analogs thereof,examples of which include those having any amino acid sequence set forthin Table 23;

Other embodiments of the inventive composition include at least onetoxin peptide (P) that is a Ptu1 peptide, and peptide analogs thereof,examples of which include those having any amino acid sequence set forthin Table 24;

Other embodiments of the inventive composition include at least onetoxin peptide (P) that is a ProTx1 peptide, and peptide analogs thereof,examples of which include those having any amino acid sequence set forthin Table 25;

Other embodiments of the inventive composition include at least onetoxin peptide (P) that is a BeKM1 peptide, and peptide analogs thereof,examples of which include those having any amino acid sequence set forthin Table 26;

Other embodiments of the inventive composition include at least onetoxin peptide (P) that is a Na⁺ channel inhibitor peptide, examples ofwhich include those having any amino acid sequence set forth in Table27;

Other embodiments of the inventive composition include at least onetoxin peptide (P) that is a Cl⁻ channel inhibitor peptide, examples ofwhich include those having any amino acid sequence set forth in Table28;

Other embodiments of the inventive composition include at least onetoxin peptide (P) that is a Kv2.1 inhibitor peptide, examples of whichinclude those having any amino acid sequence set forth in Table 29;

Other embodiments of the inventive composition include at least onetoxin peptide (P) that is a Kv4.2/Kv4.3 inhibitor peptide, examples ofwhich include those having any amino acid sequence set forth in Table30;

Other embodiments of the inventive composition include at least onetoxin peptide (P) that is a nACHR inhibitor peptide, examples of whichinclude those having any amino acid sequence set forth in Table 31; and

Other embodiments of the inventive composition include at least onetoxin peptide (P) that is an Agatoxin peptide, a peptide analog thereofor other calcium channel inhibitor peptide, examples of which includethose having any amino acid sequence set forth in Table 32.

Half-life Extending Moieties. This invention involves the presence of atleast one half-life extending moiety (F¹ and/or F² in Formula I)attached to a peptide through the N-terminus, C-terminus or a sidechainof one of the intracalary amino acid residues. Multiple half-lifeextending moieties can also be used; e.g., Fc's at each terminus or anFc at a terminus and a PEG group at the other terminus or at asidechain. In other embodiments the Fc domain can be PEGylated (e.g., inaccordance with the formulae F¹—F²-(L)_(f)-P; P-(L)_(g)-F¹—F²; orP-(L)_(g)-F¹—F²-(L)_(f)-P).

The half-life extending moiety can be selected such that the inventivecomposition achieves a sufficient hydrodynamic size to prevent clearanceby renal filtration in vivo. For example, a half-life extending moietycan be selected that is a polymeric macromolecule, which issubstantially straight chain, branched-chain, or dendritic in form.Alternatively, a half-life extending moiety can be selected such that,in vivo, the inventive composition of matter will bind to a serumprotein to form a complex, such that the complex thus formed avoidssubstantial renal clearance. The half-life extending moiety can be, forexample, a lipid; a cholesterol group (such as a steroid); acarbohydrate or oligosaccharide; or any natural or synthetic protein,polypeptide or peptide that binds to a salvage receptor.

Exemplary half-life extending moieties that can be used, in accordancewith the present invention, include an immunoglobulin Fc domain, or aportion thereof, or a biologically suitable polymer or copolymer, forexample, a polyalkylene glycol compound, such as a polyethylene glycolor a polypropylene glycol. Other appropriate polyalkylene glycolcompounds include, but are not limited to, charged or neutral polymersof the following types: dextran, polylysine, colominic acids or othercarbohydrate based polymers, polymers of amino acids, and biotinderivatives.

Other examples of the half-life extending moiety, in accordance with theinvention, include a copolymer of ethylene glycol, a copolymer ofpropylene glycol, a carboxymethylcellulose, a polyvinyl pyrrolidone, apoly-1,3-dioxolane, a poly-1,3,6-trioxane, an ethylene/maleic anhydridecopolymer, a polyaminoacid (e.g., polylysine), a dextran n-vinylpyrrolidone, a poly n-vinyl pyrrolidone, a propylene glycol homopolymer,a propylene oxide polymer, an ethylene oxide polymer, a polyoxyethylatedpolyol, a polyvinyl alcohol, a linear or branched glycosylated chain, apolyacetal, a long chain fatty acid, a long chain hydrophobic aliphaticgroup, an immunoglobulin Fc domain or a portion thereof (see, e.g.,Feige et al., Modified peptides as therapeutic agents, U.S. Pat. No.6,660,843), a CH2 domain of Fc, an albumin (e.g., human serum albumin(HSA)); see, e.g., Rosen et al., Albumin fusion proteins, U.S. Pat. No.6,926,898 and US 2005/0054051; Bridon et al., Protection of endogenoustherapeutic peptides from peptidase activity through conjugation toblood components, U.S. Pat. No. 6,887,470), a transthyretin (TTR; see,e.g., Walker et al., Use of transthyretin peptide/protein fusions toincrease the serum half-life of pharmacologically activepeptides/proteins, US 2003/0195154 A1; 2003/0191056 A1), or athyroxine-binding globulin (TBG). Thus, exemplary embodiments of theinventive compositions also include HSA fusion constructs such as butnot limited to: HSA fusions with ShK, OSK1, or modified analogs of thosetoxin peptides. Examples include HSA-L10-ShK(2-35); HSA-L1-OsK1(1-38);HSA-L10-ShK(2-35); and HSA-L10-OsK1(1-38).

Other embodiments of the half-life extending moiety, in accordance withthe invention, include peptide ligands or small (organic) moleculeligands that have binding affinity for a long half-life serum proteinunder physiological conditions of temperature, pH, and ionic strength.Examples include an albumin-binding peptide or small molecule ligand, atransthyretin-binding peptide or small molecule ligand, athyroxine-binding globulin-binding peptide or small molecule ligand, anantibody-binding peptide or small molecule ligand, or another peptide orsmall molecule that has an affinity for a long half-life serum protein.(See, e.g., Blaney et al., Method and compositions for increasing theserum half-life of pharmacologically active agents by binding totransthyretin-selective ligands, U.S. Pat. No. 5,714,142; Sato et al.,Serum albumin binding moieties, US 2003/0069395 A1; Jones et al.,Pharmaceutical active conjugates, U.S. Pat. No. 6,342,225). A “longhalf-life serum protein” is one of the hundreds of different proteinsdissolved in mammalian blood plasma, including so-called “carrierproteins” (such as albumin, transferrin and haptoglobin), fibrinogen andother blood coagulation factors, complement components, immunoglobulins,enzyme inhibitors, precursors of substances such as angiotensin andbradykinin and many other types of proteins. The invention encompassesthe use of any single species of pharmaceutically acceptable half-lifeextending moiety, such as, but not limited to, those described herein,or the use of a combination of two or more different half-life extendingmoieties, such as PEG and immunoglobulin Fc domain or a CH2 domain ofFc, albumin (e.g., HSA), an albumin-binding protein, transthyretin orTBG.

In some embodiments of the invention an Fc domain or portion thereof,such as a CH2 domain of Fc, is used as a half-life extending moiety. TheFc domain can be fused to the N-terminal (e.g., in accordance with theformula F¹-(L)_(f)-P) or C-terminal (e.g., in accordance with theformula P-(L)_(g)-F¹) of the toxin peptides or at both the N and Ctermini (e.g., in accordance with the formulae F¹-(L)_(f)-P-(L)_(g)-F²or P-(L)_(g)-F¹-(L)_(f)-P). A peptide linker sequence can be optionallyincluded between the Fc domain and the toxin peptide, as describedherein. Examples of the formula F¹-(L)_(f)-P include:Fc-L10-ShK(K22A)[2-35]; Fc-L10-ShK(R1K/K22A)[1-35];Fc-L10-ShK(R1H/K22A)[1-35]; Fc-L10-ShK(R1Q/K22A)[1-35];Fc-L10-ShK(R1Y/K22A)[1-35]; Fc-L10-PP-ShK(K22A)[1-35]; and any otherworking examples described herein. Examples of the formula P-(L)_(g)-F¹include: ShK(1-35)-L10-Fc; OsK1(1-38)-L10-Fc; Met-ShK(1-35)-L10-Fc;ShK(2-35)-L10-Fc; Gly-ShK(1-35)-L10-Fc; Osk1(1-38)-L10-Fc; and any otherworking examples described herein.

Fc variants are suitable half-life extending moieties within the scopeof this invention. A native Fc can be extensively modified to form an Fcvariant in accordance with this invention, provided binding to thesalvage receptor is maintained; see, for example WO 97/34631, WO96/32478, and WO 04/110 472. In such Fc variants, one can remove one ormore sites of a native Fc that provide structural features or functionalactivity not required by the fusion molecules of this invention. One canremove these sites by, for example, substituting or deleting residues,inserting residues into the site, or truncating portions containing thesite. The inserted or substituted residues can also be altered aminoacids, such as peptidomimetics or D-amino acids. Fc variants can bedesirable for a number of reasons, several of which are described below.Exemplary Fc variants include molecules and sequences in which:

-   1. Sites involved in disulfide bond formation are removed. Such    removal can avoid reaction with other cysteine-containing proteins    present in the host cell used to produce the molecules of the    invention. For this purpose, the cysteine-containing segment at the    N-terminus can be truncated or cysteine residues can be deleted or    substituted with other amino acids (e.g., alanyl, seryl). In    particular, one can truncate the N-terminal 20-amino acid segment of    SEQ ID NO: 2 or delete or substitute the cysteine residues at    positions 7 and 10 of SEQ ID NO: 2. Even when cysteine residues are    removed, the single chain Fc domains can still form a dimeric Fc    domain that is held together non-covalently.-   2. A native Fc is modified to make it more compatible with a    selected host cell. For example, one can remove the PA sequence near    the N-terminus of a typical native Fc, which can be recognized by a    digestive enzyme in E. coli such as proline iminopeptidase. One can    also add an N-terminal methionine residue, especially when the    molecule is expressed recombinantly in a bacterial cell such as E.    coli. The Fc domain of SEQ ID NO: 2 (FIG. 4) is one such Fc variant.-   3. A portion of the N-terminus of a native Fc is removed to prevent    N-terminal heterogeneity when expressed in a selected host cell. For    this purpose, one can delete any of the first 20 amino acid residues    at the N-terminus, particularly those at positions 1, 2, 3, 4 and 5.-   4. One or more glycosylation sites are removed. Residues that are    typically glycosylated (e.g., asparagine) can confer cytolytic    response. Such residues can be deleted or substituted with    unglycosylated residues (e.g., alanine).-   5. Sites involved in interaction with complement, such as the C1q    binding site, are removed. For example, one can delete or substitute    the EKK sequence of human IgG1. Complement recruitment may not be    advantageous for the molecules of this invention and so can be    avoided with such an Fc variant.-   6. Sites are removed that affect binding to Fc receptors other than    a salvage receptor. A native Fc can have sites for interaction with    certain white blood cells that are not required for the fusion    molecules of the present invention and so can be removed.-   7. The ADCC site is removed. ADCC sites are known in the art; see,    for example, Molec. Immunol. 29 (5): 633-9 (1992) with regard to    ADCC sites in IgG1. These sites, as well, are not required for the    fusion molecules of the present invention and so can be removed.-   8. When the native Fc is derived from a non-human antibody, the    native Fc can be humanized. Typically, to humanize a native Fc, one    will substitute selected residues in the non-human native Fc with    residues that are normally found in human native Fc. Techniques for    antibody humanization are well known in the art.

Preferred Fc variants include the following. In SEQ ID NO: 2, theleucine at position 15 can be substituted with glutamate; the glutamateat position 99, with alanine; and the lysines at positions 101 and 103,with alanines. In addition, phenyalanine residues can replace one ormore tyrosine residues.

An alternative half-life extending moiety would be a protein,polypeptide, peptide, antibody, antibody fragment, or small molecule(e.g., a peptidomimetic compound) capable of binding to a salvagereceptor. For example, one could use as a half-life extending moiety apolypeptide as described in U.S. Pat. No. 5,739,277, issued Apr. 14,1998 to Presta et al. Peptides could also be selected by phage displayfor binding to the FcRn salvage receptor. Such salvage receptor-bindingcompounds are also included within the meaning of “half-life extendingmoiety” and are within the scope of this invention. Such half-lifeextending moieties should be selected for increased half-life (e.g., byavoiding sequences recognized by proteases) and decreased immunogenicity(e.g., by favoring non-immunogenic sequences, as discovered in antibodyhumanization).

As noted above, polymer half-life extending moieties can also be usedfor F¹ and F². Various means for attaching chemical moieties useful ashalf-life extending moieties are currently available, see, e.g., PatentCooperation Treaty (“PCT”) International Publication No. WO 96/11953,entitled “N-Terminally Chemically Modified Protein Compositions andMethods,” herein incorporated by reference in its entirety. This PCTpublication discloses, among other things, the selective attachment ofwater-soluble polymers to the N-terminus of proteins.

In some embodiments of the inventive compositions, the polymer half-lifeextending moiety is polyethylene glycol (PEG), as F¹ and/or F², but itshould be understood that the inventive composition of matter, beyondpositions F¹ and/or F², can also include one or more PEGs conjugated atother sites in the molecule, such as at one or more sites on the toxinpeptide. Accordingly, some embodiments of the inventive composition ofmatter further include one or more PEG moieties conjugated to a non-PEGhalf-life extending moiety, which is F¹ and/or F², or to the toxinpeptide(s) (P), or to any combination of any of these. For example, anFc domain or portion thereof (as F1 and/or F2) in the inventivecomposition can be made mono-PEGylated, di-PEGylated, or otherwisemulti-PEGylated, by the process of reductive alkylation.

Covalent conjugation of proteins and peptides with poly(ethylene glycol)(PEG) has been widely recognized as an approach to significantly extendthe in vivo circulating half-lives of therapeutic proteins. PEGylationachieves this effect predominately by retarding renal clearance, sincethe PEG moiety adds considerable hydrodynamic radius to the protein.(Zalipsky, S., et al., Use of functionalized poly(ethylene glycol)s formodification of polypeptides., in poly(ethylene glycol) chemistry:Biotechnical and biomedical applications., J. M. Harris, Ed., PlenumPress: New York., 347-370 (1992)). Additional benefits often conferredby PEGylation of proteins and peptides include increased solubility,resistance to proteolytic degradation, and reduced immunogenicity of thetherapeutic polypeptide. The merits of protein PEGylation are evidencedby the commercialization of several PEGylated proteins includingPEG-Adenosine deaminase (Adagen™/Enzon Corp.), PEG-L-asparaginase(Oncaspar™/Enzon Corp.), PEG-Interferon α-2b(PEG-Intron™/Schering/Enzon), PEG-Interferon α-2a (PEGASYS™/Roche) andPEG-G-CSF (Neulasta™/Amgen) as well as many others in clinical trials.It

Briefly, the PEG groups are generally attached to the peptide portion ofthe composition of the invention via acylation or reductive alkylationthrough a reactive group on the PEG moiety (e.g., an aldehyde, amino,thiol, or ester group) to a reactive group on the inventive compound(e.g., an aldehyde, amino, or ester group).

A useful strategy for the PEGylation of synthetic peptides consists ofcombining, through forming a conjugate linkage in solution, a peptideand a PEG moiety, each bearing a special functionality that is mutuallyreactive toward the other. The peptides can be easily prepared withconventional solid phase synthesis (see, for example, FIGS. 5 and 6 andthe accompanying text herein). The peptides are “preactivated” with anappropriate functional group at a specific site. The precursors arepurified and fully characterized prior to reacting with the PEG moiety.Ligation of the peptide with PEG usually takes place in aqueous phaseand can be easily monitored by reverse phase analytical HPLC. ThePEGylated peptides can be easily purified by preparative HPLC andcharacterized by analytical HPLC, amino acid analysis and laserdesorption mass spectrometry.

PEG is a well-known, water soluble polymer that is commerciallyavailable or can be prepared by ring-opening polymerization of ethyleneglycol according to methods well known in the art (Sandler and Karo,Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161). Inthe present application, the term “PEG” is used broadly to encompass anypolyethylene glycol molecule, in mono-, bi-, or poly-functional form,without regard to size or to modification at an end of the PEG, and canbe represented by the formula:X—O(CH₂CH₂O)_(n-1)CH₂CH₂OH,  (X)

where n is 20 to 2300 and X is H or a terminal modification, e.g., aC₁₋₄ alkyl.

In some useful embodiments, a PEG used in the invention terminates onone end with hydroxy or methoxy, i.e., X is H or CH₃ (“methoxy PEG”). Itis noted that the other end of the PEG, which is shown in formula (II)terminating in OH, covalently attaches to an activating moiety via anether oxygen bond, an amine linkage, or amide linkage. When used in achemical structure, the term “PEG” includes the formula (II) abovewithout the hydrogen of the hydroxyl group shown, leaving the oxygenavailable to react with a free carbon atom of a linker to form an etherbond. More specifically, in order to conjugate PEG to a peptide, thepeptide must be reacted with PEG in an “activated” form. Activated PEGcan be represented by the formula:(PEG)-(A)  (XI)where PEG (defined supra) covalently attaches to a carbon atom of theactivation moiety (A) to form an ether bond, an amine linkage, or amidelinkage, and (A) contains a reactive group which can react with anamino, imino, or thiol group on an amino acid residue of a peptide or alinker moiety covalently attached to the peptide.

Techniques for the preparation of activated PEG and its conjugation tobiologically active peptides are well known in the art. (E.g., see U.S.Pat. Nos. 5,643,575, 5,919,455, 5,932,462, and 5,990,237; Thompson etal., PEGylation of polypeptides, EP 0575545 B1; Petit, Site specificprotein modification, U.S. Pat. Nos. 6,451,986, and 6,548,644; S. Hermanet al., Poly(ethylene glycol) with reactive endgroups: I. Modificationof proteins, J. Bioactive Compatible Polymers, 10:145-187 (1995); Y. Luet al., Pegylated peptides III: Solid-phase synthesis with PEGylatingreagents of varying molecular weight: synthesis of multiply PEGylatedpeptides, Reactive Polymers, 22:221-229 (1994); A. M. Felix et al.,PEGylated Peptides IV: Enhanced biological activity of site-directedPEGylated GRF analogs, Int. J. Peptide Protein Res., 46:253-264 (1995);A. M. Felix, Site-specific poly(ethylene glycol)ylation of peptides, ACSSymposium Series 680(poly(ethylene glycol)): 218-238 (1997); Y. Ikeda etal., Polyethylene glycol derivatives, their modified peptides, methodsfor producing them and use of the modified peptides, EP 0473084 B1; G.E. Means et al., Selected techniques for the modification of proteinside chains, in: Chemical modification of proteins, Holden Day, Inc.,219 (1971)).

Activated PEG, such as PEG-aldehydes or PEG-aldehyde hydrates, can bechemically synthesized by known means or obtained from commercialsources, e.g., Shearwater Polymers, (Huntsville, Ala.) or Enzon, Inc.(Piscataway, N.J.).

An example of a useful activated PEG for purposes of the presentinvention is a PEG-aldehyde compound (e.g., a methoxy PEG-aldehyde),such as PEG-propionaldehyde, which is commercially available fromShearwater Polymers (Huntsville, Ala.). PEG-propionaldehyde isrepresented by the formula PEG-CH₂CH₂CHO. (See, e.g., U.S. Pat. No.5,252,714). Other examples of useful activated PEG are PEG acetaldehydehydrate and PEG bis aldehyde hydrate, which latter yields abifunctionally activated structure. (See., e.g., Bentley et al.,Poly(ethylene glycol) aldehyde hydrates and related polymers andapplications in modifying amines, U.S. Pat. No. 5,990,237).

Another useful activated PEG for generating the PEG-conjugated peptidesof the present invention is a PEG-maleimide compound, such as, but notlimited to, a methoxy PEG-maleimide, such as maleimido monomethoxy PEG,are particularly useful for generating the PEG-conjugated peptides ofthe invention. (E.g., Shen, N-maleimidyl polymer derivatives, U.S. Pat.No. 6,602,498; C. Delgado et al., The uses and properties of PEG-linkedproteins., Crit. Rev. Therap. Drug Carrier Systems, 9:249-304 (1992); S.Zalipsky et al., Use of functionalized poly(ethylene glycol)s formodification of polypeptides, in: Poly(ethylene glycol) chemistry:Biotechnical and biomedical applications (J. M. Harris, Editor, PlenumPress: New York, 347-370 (1992); S. Herman et al., Poly(ethylene glycol)with reactive endgroups: I. Modification of proteins, J. BioactiveCompatible Polymers, 10:145-187 (1995); P. J. Shadle et al., Conjugationof polymer to colony stimulating factor-1, U.S. Pat. No. 4,847,325; G.Shaw et al., Cysteine added variants IL-3 and chemical modificationsthereof, U.S. Pat. No. 5,166,322 and EP 0469074 B1; G. Shaw et al.,Cysteine added variants of EPO and chemical modifications thereof, EP0668353 A1; G. Shaw et al., Cysteine added variants G-CSF and chemicalmodifications thereof, EP 0668354 A1; N. V. Katre et al., Interleukin-2muteins and polymer conjugation thereof, U.S. Pat. No. 5,206,344; R. J.Goodson and N. V. Katre, Site-directed pegylation of recombinantinterleukin-2 at its glycosylation site, Biotechnology, 8:343-346(1990)).

A poly(ethylene glycol) vinyl sulfone is another useful activated PEGfor generating the PEG-conjugated peptides of the present invention byconjugation at thiolated amino acid residues, e.g., at C residues.(E.g., M. Morpurgo et al., Preparation and characterization ofpoly(ethylene glycol) vinyl sulfone, Bioconj. Chem., 7:363-368 (1996);see also Harris, Functionalization of polyethylene glycol for formationof active sulfone-terminated PEG derivatives for binding to proteins andbiologically compatible materials, U.S. Pat. Nos. 5,446,090; 5,739,208;5,900,461; 6,610,281 and 6,894,025; and Harris, Water soluble activesulfones of poly(ethylene glycol), WO 95/13312 A1).

Another activated form of PEG that is useful in accordance with thepresent invention, is a PEG-N-hydroxysuccinimide ester compound, forexample, methoxy PEG-N-hydroxysuccinimidyl (NHS) ester.

Heterobifunctionally activated forms of PEG are also useful. (See, e.g.,Thompson et al., PEGylation reagents and biologically active compoundsformed therewith, U.S. Pat. No. 6,552,170).

Typically, a toxin peptide or, a fusion protein comprising the toxinpeptide, is reacted by known chemical techniques with an activated PEGcompound, such as but not limited to, a thiol-activated PEG compound, adiol-activated PEG compound, a PEG-hydrazide compound, a PEG-oxyaminecompound, or a PEG-bromoacetyl compound. (See, e.g., S. Herman,Poly(ethylene glycol) with Reactive Endgroups: I. Modification ofProteins, J. Bioactive and Compatible Polymers, 10:145-187 (1995); S.Zalipsky, Chemistry of Polyethylene Glycol Conjugates with BiologicallyActive Molecules, Advanced Drug Delivery Reviews, 16:157-182 (1995); R.Greenwald et al., Poly(ethylene glycol) conjugated drugs and prodrugs: acomprehensive review, Critical Reviews in Therapeutic Drug CarrierSystems, 17:101-161 (2000)).

Methods for N-terminal PEGylation are exemplified herein in Examples31-34, 45 and 47-48, but these are in no way limiting of the PEGylationmethods that can be employed by one skilled in the art.

Any molecular mass for a PEG can be used as practically desired, e.g.,from about 1,000 or 2,000 Daltons (Da) to about 100,000 Da (n is 20 to2300). Preferably, the combined or total molecular mass of PEG used in aPEG-conjugated peptide of the present invention is from about 3,000 Daor 5,000 Da, to about 50,000 Da or 60,000 Da (total n is from 70 to1,400), more preferably from about 10,000 Da to about 40,000 Da (total nis about 230 to about 910). The most preferred combined mass for PEG isfrom about 20,000 Da to about 30,000 Da (total n is about 450 to about680). The number of repeating units “n” in the PEG is approximated forthe molecular mass described in Daltons. It is preferred that thecombined molecular mass of PEG on an activated linker is suitable forpharmaceutical use. Thus, the combined molecular mass of the PEGmolecule should not exceed about 100,000 Da.

Polysaccharide polymers are another type of water-soluble polymer thatcan be used for protein modification. Dextrans are polysaccharidepolymers comprised of individual subunits of glucose predominantlylinked by α1-6 linkages. The dextran itself is available in manymolecular weight ranges, and is readily available in molecular weightsfrom about 1 kD to about 70 kD. Dextran is a suitable water-solublepolymer for use in the present invention as a half-life extending moietyby itself or in combination with another half-life extending moiety(e.g., Fc). See, for example, WO 96/11953 and WO 96/05309. The use ofdextran conjugated to therapeutic or diagnostic immunoglobulins has beenreported; see, for example, European Patent Publication No. 0 315 456,which is hereby incorporated by reference in its entirety. Dextran ofabout 1 kD to about 20 kD is preferred when dextran is used as ahalf-life extending moiety in accordance with the present invention.

Linkers. Any “linker” group or moiety (i.e., “-(L)_(f)-” or “-(L)_(g)-”in Formulae I-IX) is optional. When present, its chemical structure isnot critical, since it serves primarily as a spacer. As stated hereinabove, the linker moiety (-(L)_(f)- and/or -(L)_(g)-), if present, canbe independently the same or different from any other linker, orlinkers, that may be present in the inventive composition. For example,an “(L)_(f)” can represent the same moiety as, or a different moietyfrom, any other “(L)_(f)” or any “(L)_(g)” in accordance with theinvention. The linker is preferably made up of amino acids linkedtogether by peptide bonds. Thus, in some embodiments, the linker is madeup of from 1 to about 30 amino acids linked by peptide bonds, whereinthe amino acids are selected from the 20 naturally occurring aminoacids. Some of these amino acids can be glycosylated, as is wellunderstood by those in the art. For example, a useful linker sequenceconstituting a sialylation site is X₁X₂NX₄X₅G (SEQ ID NO: 637), whereinX₁, X₂,X₄ and X₅ are each independently any amino acid residue.

In some embodiments, the 1 to 20 amino acids are selected from glycine,alanine, proline, asparagine, glutamine, and lysine. Even morepreferably, a linker is made up of a majority of amino acids that aresterically unhindered, such as glycine and alanine. Thus, preferredlinkers include polyglycines (particularly (Gly)₄, (Gly)₅),poly(Gly-Ala), and polyalanines. Other preferred linkers are thoseidentified herein as “L5” (GGGGS; SEQ ID NO: 638), “L10” (GGGGSGGGGS;SEQ ID NO:79), “L25” GGGGSGGGGSGGGGSGGGGSGGGGS; SEQ ID NO:84) and anylinkers used in the working examples hereinafter. The linkers describedherein, however, are exemplary; linkers within the scope of thisinvention can be much longer and can include other residues.

In some embodiments of the compositions of this invention, whichcomprise a peptide linker moiety (L), acidic residues, for example,glutamate or aspartate residues, are placed in the amino acid sequenceof the linker moiety (L). Examples include the following peptide linkersequences:

GGEGGG; (SEQ ID NO: 639) GGEEEGGG; (SEQ ID NO: 640) GEEEG;(SEQ ID NO: 641) GEEE; (SEQ ID NO: 642) GGDGGG; (SEQ ID NO: 643)GGDDDGG; (SEQ ID NO: 644) GDDDG; (SEQ ID NO: 645) GDDD; (SEQ ID NO: 646)GGGGSDDSDEGSDGEDGGGGS; (SEQ ID NO: 647) WEWEW; (SEQ ID NO: 648) FEFEF;(SEQ ID NO: 649) EEEWWW; (SEQ ID NO: 650) EEEFFF; (SEQ ID NO: 651)WWEEEWW; (SEQ ID NO: 652) or FFEEEFF. (SEQ ID NO: 653)

In other embodiments, the linker constitutes a phosphorylation site,e.g., X₁X₂YX₃X₄G (SEQ ID NO: 654), wherein X₁, X₂, X₃ and X₄ are eachindependently any amino acid residue; X₁X₂SX₃X₄G (SEQ ID NO: 655),wherein X₁, X₂, X₃ and X₄ are each independently any amino acid residue;or X₁X₂TX₃X₄G (SEQ ID NO: 656), wherein X₁, X₂, X₃ and X₄ are eachindependently any amino acid residue.

Non-peptide linkers are also possible. For example, alkyl linkers suchas —NH—(CH₂)_(s)—C(O)—, wherein s=2-20 could be used. These alkyllinkers can further be substituted by any non-sterically hindering groupsuch as lower alkyl (e.g., C₁-C₆) lower acyl, halogen (e.g., Cl, Br),CN, NH₂, phenyl, etc. An exemplary non-peptide linker is a PEG linker,

wherein n is such that the linker has a molecular weight of 100 to 5000kD, preferably 100 to 500 kD. The peptide linkers can be altered to formderivatives in the same manner as described above.

Derivatives. The inventors also contemplate derivatizing the peptideand/or half-life extending moiety portion of the compounds. Suchderivatives can improve the solubility, absorption, biologicalhalf-life, and the like of the compounds. The moieties can alternativelyeliminate or attenuate any undesirable side-effect of the compounds andthe like. Exemplary derivatives include compounds in which:

-   1. The compound or some portion thereof is cyclic. For example, the    peptide portion can be modified to contain two or more Cys residues    (e.g., in the linker), which could cyclize by disulfide bond    formation.-   2. The compound is cross-linked or is rendered capable of    cross-linking between molecules. For example, the peptide portion    can be modified to contain one Cys residue and thereby be able to    form an intermolecular disulfide bond with a like molecule. The    compound can also be cross-linked through its C-terminus, as in the    molecule shown below.

-   3. Non-peptidyl linkages (bonds) replace one or more peptidyl    [—C(O)NR—] linkages. Exemplary non-peptidyl linkages are    —CH₂-carbamate [—CH₂—OC(O)NR—], phosphonate, —CH₂-sulfonamide    [—CH₂—S(O)₂NR—], urea [—NHC(O)NH—], —CH₂-secondary amine, and    alkylated peptide [—C(O)NR⁶— wherein R⁶ is lower alkyl].-   4. The N-terminus is chemically derivatized. Typically, the    N-terminus can be acylated or modified to a substituted amine.    Exemplary N-terminal derivative groups include —NRR¹ (other than    —NH₂), —NRC(O)R¹, —NRC(O)OR¹, —NRS(O)₂R¹, —NHC(O)NHR¹, succinimide,    or benzyloxycarbonyl-NH— (CBZ-NH—), wherein R and R¹ are each    independently hydrogen or lower alkyl and wherein the phenyl ring    can be substituted with 1 to 3 substituents selected from the group    consisting of C₁-C₄ alkyl, C₁-C₄ alkoxy, chloro, and bromo.-   5. The free C-terminus is derivatized. Typically, the C-terminus is    esterified or amidated. For example, one can use methods described    in the art to add (NH—CH₂—CH₂—NH₂)₂ to compounds of this invention    having any of SEQ ID NOS: 504 to 508 at the C-terminus. Likewise,    one can use methods described in the art to add —NH₂ to compounds of    this invention having any of SEQ ID NOS: 924 to 955, 963 to 972,    1005 to 1013, or 1018 to 1023 at the C-terminus. Exemplary    C-terminal derivative groups include, for example, —C(O)R² wherein    R² is lower alkoxy or —NR³R⁴ wherein R³ and R⁴ are independently    hydrogen or C₁-C₈ alkyl (preferably C₁-C₄ alkyl).-   6. A disulfide bond is replaced with another, preferably more    stable, cross-linking moiety (e.g., an alkylene). See, e.g.,    Bhatnagar et al. (1996), J. Med. Chem. 39: 3814-9; Alberts et    al. (1993) Thirteenth Am. Pep. Symp., 357-9.-   7. One or more individual amino acid residues are modified. Various    derivatizing agents are known to react specifically with selected    sidechains or terminal residues, as described in detail below.

Lysinyl residues and amino terminal residues can be reacted withsuccinic or other carboxylic acid anhydrides, which reverse the chargeof the lysinyl residues. Other suitable reagents for derivatizingalpha-amino-containing residues include imidoesters such as methylpicolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride;trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; andtransaminase-catalyzed reaction with glyoxylate.

Arginyl residues can be modified by reaction with any one or combinationof several conventional reagents, including phenylglyoxal,2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization ofarginyl residues requires that the reaction be performed in alkalineconditions because of the high pKa of the guanidine functional group.Furthermore, these reagents can react with the groups of lysine as wellas the arginine epsilon-amino group.

Specific modification of tyrosyl residues has been studied extensively,with particular interest in introducing spectral labels into tyrosylresidues by reaction with aromatic diazonium compounds ortetranitromethane. Most commonly, N-acetylimidizole andtetranitromethane are used to form O-acetyl tyrosyl species and 3-nitroderivatives, respectively.

Carboxyl sidechain groups (aspartyl or glutamyl) can be selectivelymodified by reaction with carbodiimides (R¹—N═C═N—R¹) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl)carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore,aspartyl and glutamyl residues can be converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues can be deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Cysteinyl residues can be replaced by amino acid residues or othermoieties either to eliminate disulfide bonding or, conversely, tostabilize cross-linking. See, e.g., Bhatnagar et al. (1996), J. Med.Chem. 39: 3814-9.

Derivatization with bifunctional agents is useful for cross-linking thepeptides or their functional derivatives to a water-insoluble supportmatrix or to other macromolecular half-life extending moieties. Commonlyused cross-linking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Carbohydrate (oligosaccharide) groups can conveniently be attached tosites that are known to be glycosylation sites in proteins. Generally,O-linked oligosaccharides are attached to serine (Ser) or threonine(Thr) residues while N-linked oligosaccharides are attached toasparagine (Asn) residues when they are part of the sequenceAsn-X-Ser/Thr, where X can be any amino acid except proline. X ispreferably one of the 19 naturally occurring amino acids other thanproline. The structures of N-linked and O-linked oligosaccharides andthe sugar residues found in each type are different. One type of sugarthat is commonly found on both is N-acetylneuraminic acid (referred toas sialic acid). Sialic acid is usually the terminal residue of bothN-linked and O-linked oligosaccharides and, by virtue of its negativecharge, can confer acidic properties to the glycosylated compound. Suchsite(s) can be incorporated in the linker of the compounds of thisinvention and are preferably glycosylated by a cell during recombinantproduction of the polypeptide compounds (e.g., in mammalian cells suchas CHO, BHK, COS). However, such sites can further be glycosylated bysynthetic or semi-synthetic procedures known in the art.

Other possible modifications include hydroxylation of proline andlysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, oxidation of the sulfur atom in Cys, methylation of thealpha-amino groups of lysine, arginine, and histidine side chains.Creighton, Proteins: Structure and Molecule Properties (W. H. Freemanand Co., San Francisco), pp. 79-86 (1983).

Compounds of the present invention can be changed at the DNA level, aswell. The DNA sequence of any portion of the compound can be changed tocodons more compatible with the chosen host cell. For E. coli, which isthe preferred host cell, optimized codons are known in the art. Codonscan be substituted to eliminate restriction sites or to include silentrestriction sites, which can aid in processing of the DNA in theselected host cell. The half-life extending moiety, linker and peptideDNA sequences can be modified to include any of the foregoing sequencechanges.

A process for preparing conjugation derivatives is also contemplated.Tumor cells, for example, exhibit epitopes not found on their normalcounterparts. Such epitopes include, for example, differentpost-translational modifications resulting from their rapidproliferation. Thus, one aspect of this invention is a processcomprising:

-   -   a) selecting at least one randomized peptide that specifically        binds to a target epitope; and    -   b) preparing a pharmacologic agent comprising (i) at least one        half-life extending moiety (Fc domain preferred), (ii) at least        one amino acid sequence of the selected peptide or peptides,        and (iii) an effector molecule.        The target epitope is preferably a tumor-specific epitope or an        epitope specific to a pathogenic organism. The effector molecule        can be any of the above-noted conjugation partners and is        preferably a radioisotope.

Methods of Making

The present invention also relates to nucleic acids, expression vectorsand host cells useful in producing the polypeptides of the presentinvention. Host cells can be eukaryotic cells, with mammalian cellspreferred and CHO cells most preferred. Host cells can also beprokaryotic cells, with E. coli cells most preferred.

The compounds of this invention largely can be made in transformed hostcells using recombinant DNA techniques. To do so, a recombinant DNAmolecule coding for the peptide is prepared. Methods of preparing suchDNA molecules are well known in the art. For instance, sequences codingfor the peptides could be excised from DNA using suitable restrictionenzymes. Alternatively, the DNA molecule could be synthesized usingchemical synthesis techniques, such as the phosphoramidate method. Also,a combination of these techniques could be used

The invention also includes a vector capable of expressing the peptidesin an appropriate host. The vector comprises the DNA molecule that codesfor the peptides operatively linked to appropriate expression controlsequences. Methods of effecting this operative linking, either before orafter the DNA molecule is inserted into the vector, are well known.Expression control sequences include promoters, activators, enhancers,operators, ribosomal binding sites, start signals, stop signals, capsignals, polyadenylation signals, and other signals involved with thecontrol of transcription or translation.

The resulting vector having the DNA molecule thereon is used totransform an appropriate host. This transformation can be performedusing methods well known in the art.

Any of a large number of available and well-known host cells can be usedin the practice of this invention. The selection of a particular host isdependent upon a number of factors recognized by the art. These include,for example, compatibility with the chosen expression vector, toxicityof the peptides encoded by the DNA molecule, rate of transformation,ease of recovery of the peptides, expression characteristics, bio-safetyand costs. A balance of these factors must be struck with theunderstanding that not all hosts can be equally effective for theexpression of a particular DNA sequence. Within these generalguidelines, useful microbial hosts include bacteria (such as E. colisp.), yeast (such as Saccharomyces sp.) and other fungi, insects,plants, mammalian (including human) cells in culture, or other hostsknown in the art.

Next, the transformed host is cultured and purified. Host cells can becultured under conventional fermentation conditions so that the desiredcompounds are expressed. Such fermentation conditions are well known inthe art. Finally, the peptides are purified from culture by methods wellknown in the art.

The compounds can also be made by synthetic methods. Solid phasesynthesis is the preferred technique of making individual peptides sinceit is the most cost-effective method of making small peptides. Forexample, well known solid phase synthesis techniques include the use ofprotecting groups, linkers, and solid phase supports, as well asspecific protection and deprotection reaction conditions, linkercleavage conditions, use of scavengers, and other aspects of solid phasepeptide synthesis. Suitable techniques are well known in the art. (E.g.,Merrifield (1973), Chem. Polypeptides, pp. 335-61 (Katsoyannis andPanayotis eds.); Merrifield (1963), J. Am. Chem. Soc. 85: 2149; Davis etal. (1985), Biochem. Intl. 10: 394-414; Stewart and Young (1969), SolidPhase Peptide Synthesis; U.S. Pat. No. 3,941,763; Finn et al. (1976),The Proteins (3rd ed.) 2: 105-253; and Erickson et al. (1976), TheProteins (3rd ed.) 2: 257-527; “Protecting Groups in Organic Synthesis,”3rd Edition, T. W. Greene and P. G. M. Wuts, Eds., John Wiley & Sons,Inc., 1999; NovaBiochem Catalog, 2000; “Synthetic Peptides, A User'sGuide,” G. A. Grant, Ed., W. H. Freeman & Company, New York, N.Y., 1992;“Advanced Chemtech Handbook of Combinatorial & Solid Phase OrganicChemistry,” W. D. Bennet, J. W. Christensen, L. K. Hamaker, M. L.Peterson, M. R. Rhodes, and H. H. Saneii, Eds., Advanced Chemtech, 1998;“Principles of Peptide Synthesis, 2nd ed.,” M. Bodanszky, Ed.,Springer-Verlag, 1993; “The Practice of Peptide Synthesis, 2nd ed.,” M.Bodanszky and A. Bodanszky, Eds., Springer-Verlag, 1994; “ProtectingGroups,” P. J. Kocienski, Ed., Georg Thieme Verlag, Stuttgart, Germany,1994; “Fmoc Solid Phase Peptide Synthesis, A Practical Approach,” W. C.Chan and P. D. White, Eds., Oxford Press, 2000, G. B. Fields et al.,Synthetic Peptides: A User's Guide, 1990, 77-183).

Whether the compositions of the present invention are prepared bysynthetic or recombinant techniques, suitable protein purificationtechniques can also be involved, when applicable. In some embodiments ofthe compositions of the invention, the toxin peptide portion and/or thehalf-life extending portion, or any other portion, can be prepared toinclude a suitable isotopic label (e.g., ¹²⁵I, ¹⁴C, ¹³C, ³⁵S, ³H, ²H,¹³N, ¹⁵N, ¹⁸O, ¹⁷O, etc.), for ease of quantification or detection.

Compounds that contain derivatized peptides or which contain non-peptidegroups can be synthesized by well-known organic chemistry techniques.

Uses of the Compounds

In General. The compounds of this invention have pharmacologic activityresulting from their ability to bind to proteins of interest asagonists, mimetics or antagonists of the native ligands of such proteinsof interest. Heritable diseases that have a known linkage to ionchannels (“channelopathies”) cover various fields of medicine, some ofwhich include neurology, nephrology, myology and cardiology. A list ofinherited disorders attributed to ion channels includes:

-   -   cystic fibrosis (Cl⁻ channel; CFTR),    -   Dent's disease (proteinuria and hypercalciuria; Cl⁻ channel;        CLCN5),    -   osteopetrosis (Cl⁻ channel; CLCN7),    -   familial hyperinsulinemia (SUR1; KCNJ11; K channel),    -   diabetes (KATP/SUR channel),    -   Andersen syndrome (KCNJ2, Kir2.1 K channel),    -   Bartter syndrome (KCNJ1; Kir1.1/ROMK; K channel),    -   hereditary hearing loss (KCNQ4; K channel),    -   hereditary hypertension (Liddle's syndrome; SCNN1; epithelial Na        channel),    -   dilated cardiomyopathy (SUR2, K channel),    -   long-QT syndrome or cardiac arrhythmias (cardiac potassium and        sodium channels),    -   Thymothy syndrome (CACNA1C, Cav1.2),    -   myasthenic syndromes (CHRNA, CHRNB, CNRNE; nAChR), and a variety        of other myopathies,    -   hyperkalemic periodic paralysis (Na and K channels),    -   epilepsy (Na⁺ and K⁺ channels),    -   hemiplegic migraine (CACNA1A, Cav2.1 Ca²⁺ channel and ATP1A2),    -   central core disease (RYR1, RyR1; Ca²⁺ channel), and    -   paramyotonia and myotonia (Na⁺, Cl⁻ channels)        See L. J. Ptacek and Y-H Fu (2004), Arch. Neurol. 61:        166-8; B. A. Niemeyer et al. (2001), EMBO reports 21: 568-73; F.        Lehmann-Horn and K. Jurkat-Rott (1999), Physiol. Rev. 79:        1317-72. Although the foregoing list concerned disorders of        inherited origin, molecules targeting the channels cited in        these disorders can also be useful in treating related disorders        of other, or indeterminate, origin.

In addition to the aforementioned disorders, evidence has also beenprovided supporting ion channels as targets for treatment of:

-   -   sickle cell anemia (IKCa1)—in sickle cell anemia, water loss        from erythrocytes leads to hemoglobin polymerization and        subsequent hemolysis and vascular obstruction. The water loss is        consequent to potassium efflux through the so-called Gardos        channel i.e., IKCa1. Therefore, block of IKCa1 is a potential        therapeutic treatment for sickle cell anemia.    -   glaucoma (BKCa),—in glaucoma the intraocular pressure is too        high leading to optic nerve damage, abnormal eye function and        possibly blindness. Block of BKCa potassium channels can reduce        intraocular fluid secretion and increase smooth muscle        contraction, possibly leading to lower intraocular pressure and        neuroprotection in the eye.    -   multiple sclerosis (Kv, KCa),    -   psoriasis (Kv, KCa),    -   arthritis (Kv, KCa),    -   asthma (KCa, Kv),    -   allergy (KCa, Kv),    -   COPD (KCa, Kv, Ca),    -   allergic rhinitis (KCa, Kv),    -   pulmonary fibrosis,    -   lupus (IKCa1, Kv),    -   transplantation, GvHD (KCa, Kv),    -   inflammatory bone resorption (KCa, Kv),    -   periodontal disease (KCa, Kv),    -   diabetes, type I (Kv),—type I diabetes is an autoimmune disease        that is characterized by abnormal glucose, protein and lipid        metabolism and is associated with insulin deficiency or        resistance. In this disease, Kv1.3-expressing T-lymphocytes        attack and destroy pancreatic islets leading to loss of        beta-cells. Block of Kv1.3 decreases inflammatory cytokines. In        addition block of Kv1.3 facilitates the translocation of GLUT4        to the plasma membrane, thereby increasing insulin sensitivity.    -   obesity (Kv),—Kv1.3 appears to play a critical role in        controlling energy homeostasis and in protecting against        diet-induced obesity. Consequently, Kv1.3 blockers could        increase metabolic rate, leading to greater energy utilization        and decreased body weight.    -   restenosis (KCa, Ca²⁺),—proliferation and migration of vascular        smooth muscle cells can lead to neointimal thickening and        vascular restenosis. Excessive neointimal vascular smooth muscle        cell proliferation is associated with elevated expression of        IKCa1. Therefore, block of IKCa1 could represent a therapeutic        strategy to prevent restenosis after angioplasty.    -   ischaemia (KCa, Ca²⁺),—in neuronal or cardiac ischemia,        depolarization of cell membranes leads to opening of        voltage-gated sodium and calcium channels. In turn this can lead        to calcium overload, which is cytotoxic. Block of voltage-gated        sodium and/or calcium channels can reduce calcium overload and        provide cytoprotective effects. In addition, due to their        critical role in controlling and stabilizing cell membrane        potential, modulators of voltage- and calcium-activated        potassium channels can also act to reduce calcium overload and        protect cells.    -   renal incontinence (KCa), renal incontinence is associated with        overactive bladder smooth muscle cells. Calcium-activated        potassium channels are expressed in bladder smooth muscle cells,        where they control the membrane potential and indirectly control        the force and frequency of cell contraction. Openers of        calcium-activated potassium channels therefore provide a        mechanism to dampen electrical and contractile activity in        bladder, leading to reduced urge to urinate.    -   osteoporosis (Kv),    -   pain, including migraine (Na_(v), TRP [transient receptor        potential channels], P2X, Ca²⁺), N-type voltage-gated calcium        channels are key regulators of nociceptive neurotransmission in        the spinal cord. Ziconotide, a peptide blocker of N-type calcium        channels reduces nociceptive neurotransmission and is approved        worldwide for the symptomatic alleviation of severe chronic pain        in humans. Novel blockers of nociceptor-specific N-type calcium        channels would be improved analgesics with reduced side-effect        profiles.    -   hypertension (Ca²⁺),—L-type and T-type voltage-gated calcium        channels are expressed in vascular smooth muscle cells where        they control excitation-contraction coupling and cellular        proliferation. In particular, T-type calcium channel activity        has been linked to neointima formation during hypertension.        Blockers of L-type and T-type calcium channels are useful for        the clinical treatment of hypertension because they reduce        calcium influx and inhibit smooth muscle cell contraction.    -   wound healing, cell migration serves a key role in wound        healing. Intracellular calcium gradients have been implicated as        important regulators of cellular migration machinery in        keratinocytes and fibroblasts. In addition, ion flux across cell        membranes is associated with cell volume changes. By controlling        cell volume, ion channels contribute to the intracellular        environment that is required for operation of the cellular        migration machinery. In particular, IKCa1 appears to be required        universally for cell migration. In addition, Kv1.3, Kv3.1, NMDA        receptors and N-type calcium channels are associated with the        migration of lymphocytes and neurons.    -   stroke,    -   Alzheimer's, Parkenson's Disease (nACHR, Nav) Bipolar Disorder        (Nav, Cav)    -   cancer, many potassium channel genes are amplified and protein        subunits are upregulated in many cancerous condition. Consistent        with a pathophysiological role for potassium channel        upregulation, potassium channel blockers have been shown to        suppress proliferation of uterine cancer cells and        hepatocarcinoma cells, presumably through inhibition of calcium        influx and effects on calcium-dependent gene expression.    -   a variety of neurological, cardiovascular, metabolic and        autoimmune diseases.

Both agonists and antagonists of ion channels can achieve therapeuticbenefit. Therapeutic benefits can result, for example, from antagonizingKv1.3, IKCa1, SKCa, BKCa, N-type or T-type Ca²⁺ channels and the like.Small molecule and peptide antagonists of these channels have been shownto possess utility in vitro and in vivo. Limitations in productionefficiency and pharmacokinetics, however, have largely preventedclinical investigation of inhibitor peptides of ion channels.

Compositions of this invention incorporating peptide antagonists of thevoltage-gated potassium channel Kv1.3 are useful as immunosuppressiveagents with therapeutic value for autoimmune diseases. For example, suchmolecules are useful in treating multiple sclerosis, type 1 diabetes,psoriasis, inflammatory bowel disease, and rheumatoid arthritis. (See,e.g., H. Wulff et al. (2003) J. Clin. Invest. 111, 1703-1713 and H. Ruset al. (2005) PNAS 102, 11094-11099; Beeton et al., Targeting effectormemory T cells with a selective inhibitor peptide of Kv1.3 channnels fortherapy of autoimmune diseases, Molec. Pharmacol. 67(4):1369-81 (2005);1 Beeton et al. (2006), Kv1.3: therapeutic target for cell-mediatedautoimmune disease, electronic preprint at//webfiles.uci.edu/xythoswfs/webui/2670029.1). Inhibitors of thevoltage-gated potassium channel Kv1.3 have been examined in a variety ofpreclinical animal models of inflammation. Small molecule and peptideinhibitors of Kv1.3 have been shown to block delayed typehypersensitivity responses to ovalbumin [C. Beeton et al. (2005) Mol.Pharmacol. 67, 1369] and tetanus toxoid [G. C. Koo et al. (1999) Clin.Immunol. 197, 99]. In addition to suppressing inflammation in the skin,inhibitors also reduced antibody production [G. C. Koo et al. (1997) J.Immunol. 158, 5120]. Kv1.3 antagonists have shown efficacy in a ratadoptive-transfer experimental autoimmune encephalomyelitis (AT-EAE)model of multiple sclerosis (MS). The Kv1.3 channel is overexpressed onmyelin-specific T cells from MS patients, lending further support to theutility Kv1.3 inhibitors may provide in treating MS. Inflammatory boneresorption was also suppressed by Kv1.3 inhibitors in a preclinicaladoptive-transfer model of periodontal disease [P. Valverde et al.(2004) J. Bone Mineral Res. 19, 155]. In this study, inhibitorsadditionally blocked antibody production to a bacterial outer membraneprotein,—one component of the bacteria used to induce gingivalinflammation. Recently in preclinical rat models, efficacy of Kv1.3inhibitors was shown in treating pristane-induced arthritis and diabetes[C. Beeton et al. (2006) preprint available at//webfiles.uci.edu/xythoswfs/webui/_xy-2670029_(—)1.]. The Kv1.3 channelis expressed on all subsets of T cells and B cells, but effector memoryT cells and class-switched memory B cells are particularly dependent onKv1.3 [H. Wulff et al. (2004) J. Immunol. 173, 776].Gad5/insulin-specific T cells from patients with new onset type 1diabetes, myelin-specific T cells from MS patients and T cells from thesynovium of rheumatoid arthritis patients all overexpress Kv1.3 [C.Beeton et al. (2006) preprint at//webfiles.uci.edu/xythoswfs/webui/_xy-2670029_(—)1.]. Because micedeficient in Kv1.3 gained less weight when placed on a high fat diet [J.Xu et al. (2003) Human Mol. Genet. 12, 551] and showed altered glucoseutilization [J. Xu et al. (2004) Proc. Natl. Acad. Sci. 101, 3112],Kv1.3 is also being investigated for the treatment of obesity anddiabetes. Breast cancer specimens [M. Abdul et al. (2003) AnticancerRes. 23, 3347] and prostate cancer cell lines [S. P. Fraser et al.(2003) Pflugers Arch. 446, 559] have also been shown to express Kv1.3,and Kv1.3 blockade may be of utility for treatment of cancer. Disordersthat can be treated in accordance with the inventive method of treatingan autoimmune disorder, involving Kv1.3 inhibitor toxin peptide(s),include multiple sclerosis, type 1 diabetes, psoriasis, inflammatorybowel disease, contact-mediated dermatitis, rheumatoid arthritis,psoriatic arthritis, asthma, allergy, restinosis, systemic sclerosis,fibrosis, scleroderma, glomerulonephritis, Sjogren syndrome,inflammatory bone resorption, transplant rejection, graft-versus-hostdisease, and systemic lupus erythematosus (SLE) and other forms oflupus.

Some of the cells that express the calcium-activated potassium ofintermediate conductance IKCa1 include T cells, B cells, mast cells andred blood cells (RBCs). T cells and RBCs from mice deficient in IKCa1show defects in volume regulation [T. Begenisich et al. (2004) J. Biol.Chem. 279, 47681]. Preclinical and clinical studies have demonstratedIKCa1 inhibitors utility in treating sickle cell anemia [J. W. Stockeret al. (2003) Blood 101, 2412; www.icagen.com]. Blockers of the IKCa1channel have also been shown to block EAE, indicating they may possessutility in treatment of MS [E. P. Reich et al. (2005) Eur. J. Immunol.35, 1027]. IgE-mediated histamine production from mast cells is alsoblocked by IKCa1 inhibitors [S. Mark Duffy et al. (2004) J. AllergyClin. Immunol. 114, 66], therefore they may also be of benefit intreating asthma. The IKCa1 channel is overexpressed on activated T and Blymphocytes [H. Wulff et al. (2004) J. Immunol. 173, 776] and thus mayshow utility in treatment of a wide variety of immune disorders. Outsideof the immune system, IKCa1 inhibitors have also shown efficacy in a ratmodel of vascular restinosis and thus might represent a new therapeuticstrategy to prevent restenosis after angioplasty [R. Kohler et al.(2003) Circulation 108, 1119]. It is also thought that IKCa1 antagonistsare of utility in treatment of tumor angiogenesis since inhibitorssuppressed endothelial cell proliferation and angionenesis in vivo [I.Grgic et al. (2005) Arterioscler. Thromb. Vasc. Biol. 25, 704]. TheIKCa1 channel is upregulated in pancreatic tumors and inhibitors blockedproliferation of pancreatic tumor cell lines [H. Jager et al. (2004) MolPharmacol. 65, 630]. IKCa1 antagonists may also represent an approach toattenuate acute brain damage caused by traumatic brain injury [F. Mauler(2004) Eur. J. Neurosci. 20, 1761]. Disorders that can be treated withIKCa1 inhibitors include multiple sclerosis, asthma, psoriasis,contact-mediated dermatitis, rheumatoid & psoriatic arthritis,inflammatory bowel disease, transplant rejection, graft-versus-hostdisease, Lupus, restinosis, pancreatic cancer, tumor angiogenesis andtraumatic brain injury.

Accordingly, molecules of this invention incorporating peptideantagonists of the calcium-activated potassium channel of intermediateconductance, IKCa can be used to treat immune dysfunction, multiplesclerosis, type 1 diabetes, psoriasis, inflammatory bowel disease,contact-mediated dermatitis, rheumatoid arthritis, psoriatic arthritis,asthma, allergy, restinosis, systemic sclerosis, fibrosis, scleroderma,glomerulonephritis, Sjogren syndrome, inflammatory bone resorption,transplant rejection, graft-versus-host disease, and lupus.

Accordingly, the present invention includes a method of treating anautoimmune disorder, which involves administering to a patient who hasbeen diagnosed with an autoimmune disorder, such as multiple sclerosis,type 1 diabetes, psoriasis, inflammatory bowel disease, contact-mediateddermatitis, rheumatoid arthritis, psoriatic arthritis, asthma, allergy,restinosis, systemic sclerosis, fibrosis, scleroderma,glomerulonephritis, Sjogren syndrome, inflammatory bone resorption,transplant rejection, graft-versus-host disease, or lupus, atherapeutically effective amount of the inventive composition of matter,whereby at least one symptom of the disorder is alleviated in thepatient. “Alleviated” means to be lessened, lightened, diminished,softened, mitigated (i.e., made more mild or gentle), quieted, assuaged,abated, relieved, nullified, or allayed, regardless of whether thesymptom of interest is entirely erased, eradicated, eliminated, orprevented in a particular patient.

The present invention is further directed to a method of preventing ormitigating a relapse of a symptom of multiple sclerosis, which methodinvolves administering to a patient, who has previously experienced atleast one symptom of multiple sclerosis, a prophylactically effectiveamount of the inventive composition of matter, such that the at leastone symptom of multiple sclerosis is prevented from recurring or ismitigated.

The inventive compositions of matter preferred for use in practicing theinventive method of treating an autoimmune disorder and the method ofpreventing or mitigating a relapse of a symptom of multiple sclerosisinclude as P (conjugated as in Formula I), a Kv1.3 or IKCa1 antagonistpeptide, such as a ShK peptide, an OSK1 peptide, a ChTx peptide and/or aMaurotoxin (MTx) peptide, or peptide analogs of any of these.

For example, the conjugated ShK peptide peptide or ShK peptide analogcan comprise an amino acid sequence selected from the following:

-   SEQ ID NOS: 5, 88 through 200, 548 through 561, 884 through 950, or    1295 through 1300 as set forth in Table 2.

The conjugated OSK1 peptide peptide or OSK1 peptide analog can comprisean amino acid sequence selected from the following:

-   SEQ ID NOS: 25, 294 through 298, 562 through 636, 980 through 1274,-   GVIINVSCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK (OSK1-S7) (SEQ ID NO: 1303),    or-   GVIINVSCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK (OSK1-S7,K16,D20) (SEQ ID    NO: 1308) as set forth in Table 7.

Also by way of example, a the conjugated MTX peptide, MTX peptideanalog, ChTx peptide or ChTx peptide analog can comprise an amino acidsequence selected from:

-   SEQ ID NOS: 20, 330 through 343, 1301, 1302, 1304 through 1307,    1309, 1311, 1312, or 1315 through 1336 as set forth in Table 13; or    SEQ ID NOS: 36, 59, 344 through 346, or 1369 through 1390 as set    forth in Table 14.

Also useful in these methods conjugated, or unconjugated, are a Kv1.3 orIKCa1 inhibitor toxin peptide analog that comprises an amino acidsequence selected from:

-   SEQ ID NOS: 88, 89, 92, 148 through 200, 548 through 561, 884    through 949, or 1295 through 1300 as set forth in Table 2; or-   SEQ ID NOS: 980 through 1274, GVIINVSCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK    (OSK1-S7) (SEQ ID NO: 1303), or    GVIINVSCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK (OSK1-S7,K16,D20) (SEQ ID    NO: 1308) as set forth in Table 7; or-   SEQ ID NOS: 330 through 337, 341, 1301, 1302, 1304 through 1307,    1309, 1311, 1312, and 1315 through 1336 as set forth in Table 13.

In accordance with these inventive methods, a patient who has beendiagnosed with an autoimmune disorder, such as, but not limited tomultiple sclerosis, type 1 diabetes, psoriasis, inflammatory boweldisease, contact-mediated dermatitis, rheumatoid arthritis, psoriaticarthritis, asthma, allergy, restinosis, systemic sclerosis, fibrosis,scleroderma, glomerulonephritis, Sjogren syndrome, inflammatory boneresorption, transplant rejection, graft-versus-host disease, or lupus,or a patient who has previously experienced at least one symptom ofmultiple sclerosis, are well-recognizable and/or diagnosed by theskilled practitioner, such as a physician, familiar with autoimmunedisorders and their symptoms.

For example, symptoms of multiple sclerosis can include the following:

visual symptoms, such as, optic neuritis (blurred vision, eye pain, lossof color vision, blindness); diplopia (double vision); nystagmus (jerkyeye movements); ocular dysmetria (constant under- or overshooting eyemovements); internuclear ophthalmoplegia (lack of coordination betweenthe two eyes, nystagmus, diplopia); movement and sound phosphenes(flashing lights when moving eyes or in response to a sudden noise);afferent pupillary defect (abnormal pupil responses);

motor symptoms, such as, paresis, monoparesis, paraparesis, hemiparesis,quadraparesis (muscle weakness—partial or mild paralysis); plegia,paraplegia, hemiplegia, tetraplegia, quadraplegia (paralysis—total ornear total loss of muscle strength); spasticity (loss of muscle tonecausing stiffness, pain and restricting free movement of affectedlimbs); dysarthria (slurred speech and related speech problems); muscleatrophy (wasting of muscles due to lack of use); spasms, cramps(involuntary contraction of muscles); hypotonia, clonus (problems withposture); myoclonus, myokymia (jerking and twitching muscles, tics);restless leg syndrome (involuntary leg movements, especially bothersomeat night); footdrop (foot drags along floor during walking);dysfunctional reflexes (MSRs, Babinski's, Hoffman's, Chaddock's);

sensory symptoms, such as, paraesthesia (partial numbness, tingling,buzzing and vibration sensations); anaesthesia (complete numbness/lossof sensation); neuralgia, neuropathic and neurogenic pain (pain withoutapparent cause, burning, itching and electrical shock sensations);L'Hermitte's (electric shocks and buzzing sensations when moving head);proprioceptive dysfunction (loss of awareness of location of bodyparts); trigeminal neuralgia (facial pain);

coordination and balance symptoms, such as, ataxia (loss ofcoordination); intention tremor (shaking when performing finemovements); dysmetria (constant under- or overshooting limb movements);vestibular ataxia (abnormal balance function in the inner ear); vertigo(nausea/vomitting/sensitivity to travel sickness from vestibularataxia); speech ataxia (problems coordinating speech, stuttering);dystonia (slow limb position feedback); dysdiadochokinesia (loss ofability to produce rapidly alternating movements, for example to move toa rhythm);

bowel, bladder and sexual symptoms, such as, frequent micturation,bladder spasticity (urinary urgency and incontinence); flaccid bladder,detrusor-sphincter dyssynergia (urinary hesitancy and retention);erectile dysfunction (male and female impotence); anorgasmy (inabilityto achieve orgasm); retrograde ejaculation (ejaculating into thebladder); frigidity (inability to become sexually aroused); constipation(infrequent or irregular bowel movements); fecal urgency (bowelurgency); fecal incontinence (bowel incontinence);

cognitive symptoms, such as, depression; cognitive dysfunction(short-term and long-term memory problems, forgetfulness, slow wordrecall); dementia; mood swings, emotional lability, euphoria; bipolarsyndrome; anxiety; aphasia, dysphasia (impairments to speechcomprehension and production); and

other symptoms, such as, fatigue; Uhthoff's Symptom (increase inseverity of symptoms with heat); gastroesophageal reflux (acid reflux);impaired sense of taste and smell; epileptic seizures; swallowingproblems, respiratory problems; and sleeping disorders.

The symptoms of multiple sclerosis enumerated above, are merelyillustrative and are not intended to be an exhaustive description of allpossible symptoms experienced by a single patient or by severalsufferers in composite, and to which the present invention is directed.Those skilled in the art are aware of various clinical symptoms andconstellations of symptoms of autoimmune disorders suffered byindividual patients, and to those symptoms are also directed the presentinventive methods of treating an autoimmune disorder or of preventing ormitigating a relapse of a symptom of multiple sclerosis.

The therapeutically effective amount, prophylactically effective amount,and dosage regimen involved in the inventive methods of treating anautoimmune disorder or of preventing or mitigating a relapse of asymptom of multiple sclerosis, will be determined by the attendingphysician, considering various factors which modify the action oftherapeutic agents, such as the age, condition, body weight, sex anddiet of the patient, the severity of the condition being treated, timeof administration, and other clinical factors. Generally, the dailyamount or regimen should be in the range of about 1 to about 10,000micrograms (μg) of the vehicle-conjugated peptide per kilogram (kg) ofbody mass, preferably about 1 to about 5000 μg per kilogram of bodymass, and most preferably about 1 to about 1000 μg per kilogram of bodymass.

Molecules of this invention incorporating peptide antagonists of thevoltage-gated potassium channel Kv2.1 can be used to treat type IIdiabetes.

Molecules of this invention incorporating peptide antagonists of the Mcurrent (e.g., BeKm-1) can be used to treat Alzheimer's disease andenhance cognition.

Molecules of this invention incorporating peptide antagonists of thevoltage-gated potassium channel Kv4.3 can be used to treat Alzheimer'sdisease.

Molecules of this invention incorporating peptide antagonists of thecalcium-activated potassium channel of small conductance, SKCa can beused to treat epilepsy, memory, learning, neuropsychiatric,neurological, neuromuscular, and immunological disorders, schizophrenia,bipolar disorder, sleep apnea, neurodegeneration, and smooth muscledisorders.

Molecules of this invention incorporating N-type calcium channelantagonist peptides are useful in alleviating pain. Peptides with suchactivity (e.g., Ziconotide™, ω-conotoxin-MVIIA) have been clinicallyvalidated.

Molecules of this invention incorporating T-type calcium channelantagonist peptides are useful in alleviating pain. Several lines ofevidence have converged to indicate that inhibition of Cav3.2 in dorsalroot ganglia may bring relief from chronic pain. T-type calcium channelsare found at extremely high levels in the cell bodies of a subset ofneurons in the DRG; these are likely mechanoreceptors adapted to detectslowly-moving stimuli (Shin et al., Nature Neuroscience 6:724-730,2003), and T-type channel activity is likely responsible for burstspiking (Nelson et al., J Neurosci 25:8766-8775, 2005). Inhibition ofT-type channels by either mibefradil or ethosuximide reverses mechanicalallodynia in animals induced by nerve injury (Dogrul et al., Pain105:159-168, 2003) or by chemotherapy (Flatters and Bennett, Pain109:150-161, 2004). Antisense to Cav3.2, but not Cav3.1 or Cav3.3,increases pain thresholds in animals and also reduces expression ofCav3.2 protein in the DRG (Bourinet et al., EMBO J 24:315-324, 2005).Similarly, locally injected reducing agents produce pain and increaseCav3.2 currents, oxidizing agents reduce pain and inhibit Cav3.2currents, and peripherally administered neurosteroids are analgesic andinhibit T-type currents from DRG (Todorovic et al., Pain 109:328-339,2004; Pathirathna et al., Pain 114:429-443, 2005). Accordingly, it isthought that inhibition of Cav3.2 in the cell bodies of DRG neurons caninhibit the repetitive spiking of these neurons associated with chronicpain states.

Molecules of this invention incorporating L-type calcium channelantagonist peptides are useful in treating hypertension. Small moleculeswith such activity (e.g., DHP) have been clinically validated.

Molecules of this invention incorporating peptide antagonists of theNa_(v)1 (TTX_(S)-type) channel can be used to alleviate pain. Localanesthetics and tricyclic antidepressants with such activity have beenclinically validated. Such molecules of this invention can in particularbe useful as muscle relaxants.

Molecules of this invention incorporating peptide antagonists of theNa_(v)1 (TTX_(R)-type) channel can be used to alleviate pain arisingfrom nerve and or tissue injury.

Molecules of this invention incorporating peptide antagonists of glial &epithelial cell Ca²⁺-activated chloride channel can be used to treatcancer and diabetes.

Molecules of this invention incorporating peptide antagonists of NMDAreceptors can be used to treat pain, epilepsy, brain and spinal cordinjury.

Molecules of this invention incorporating peptide antagonists ofnicotinic receptors can be used as muscle relaxants. Such molecules canbe used to treat pain, gastric motility disorders, urinary incontinence,nicotine addiction, and mood disorders.

Molecules of this invention incorporating peptide antagonists of 5HT3receptor can be used to treat Nausea, pain, and anxiety.

Molecules of this invention incorporating peptide antagonists of thenorepinephrine transporter can be used to treat pain, anti-depressant,learning, memory, and urinary incontinence.

Molecules of this invention incorporating peptide antagonists of theNeurotensin receptor can be used to treat pain.

In addition to therapeutic uses, the compounds of the present inventioncan be useful in diagnosing diseases characterized by dysfunction oftheir associated protein of interest. In one embodiment, a method ofdetecting in a biological sample a protein of interest (e.g., areceptor) that is capable of being activated comprising the steps of:(a) contacting the sample with a compound of this invention; and (b)detecting activation of the protein of interest by the compound. Thebiological samples include tissue specimens, intact cells, or extractsthereof. The compounds of this invention can be used as part of adiagnostic kit to detect the presence of their associated proteins ofinterest in a biological sample. Such kits employ the compounds of theinvention having an attached label to allow for detection. The compoundsare useful for identifying normal or abnormal proteins of interest.

The therapeutic methods, compositions and compounds of the presentinvention can also be employed, alone or in combination with othermolecules in the treatment of disease.

Pharmaceutical Compositions

In General. The present invention also provides pharmaceuticalcompositions comprising the inventive composition of matter and apharmaceutically acceptable carrier. Such pharmaceutical compositionscan be configured for administration to a patient by a wide variety ofdelivery routes, e.g., an intravascular delivery route such as byinjection or infusion, subcutaneous, intramuscular, intraperitoneal,epidural, or intrathecal delivery routes, or for oral, enteral,pulmonary (e.g., inhalant), intranasal, transmucosal (e.g., sublingualadministration), transdermal or other delivery routes and/or forms ofadministration known in the art. The inventive pharmaceuticalcompositions may be prepared in liquid form, or may be in dried powderform, such as lyophilized form. For oral or enteral use, thepharmaceutical compositions can be configured, for example, as tablets,troches, lozenges, aqueous or oily suspensions, dispersible powders orgranules, emulsions, hard or soft capsules, syrups, elixirs or enteralformulas.

In the practice of this invention the “pharmaceutically acceptablecarrier” is any physiologically tolerated substance known to those ofordinary skill in the art useful in formulating pharmaceuticalcompositions, including, any pharmaceutically acceptable diluents,excipients, dispersants, binders, fillers, glidants, anti-frictionalagents, compression aids, tablet-disintegrating agents (disintegrants),suspending agents, lubricants, flavorants, odorants, sweeteners,permeation or penetration enhancers, preservatives, surfactants,solubilizers, emulsifiers, thickeners, adjuvants, dyes, coatings,encapsulating material(s), and/or other additives singly or incombination. Such pharmaceutical compositions can include diluents ofvarious buffer content (e.g., Tris-HCl, acetate, phosphate), pH andionic strength; additives such as detergents and solubilizing agents(e.g., Tween® 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid,sodium metabisulfite), preservatives (e.g., Thimersol®, benzyl alcohol)and bulking substances (e.g., lactose, mannitol); incorporation of thematerial into particulate preparations of polymeric compounds such aspolylactic acid, polyglycolic acid, etc. or into liposomes. Hyaluronicacid can also be used, and this can have the effect of promotingsustained duration in the circulation. Such compositions can influencethe physical state, stability, rate of in vivo release, and rate of invivo clearance of the present proteins and derivatives. See, e.g.,Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack PublishingCo., Easton, Pa. 18042) pages 1435-1712, which are herein incorporatedby reference in their entirety. The compositions can be prepared inliquid form, or can be in dried powder, such as lyophilized form.Implantable sustained release formulations are also useful, as aretransdermal or transmucosal formulations. Additionally (oralternatively), the present invention provides compositions for use inany of the various slow or sustained release formulations ormicroparticle formulations known to the skilled artisan, for example,sustained release microparticle formulations, which can be administeredvia pulmonary, intranasal, or subcutaneous delivery routes.

One can dilute the inventive compositions or increase the volume of thepharmaceutical compositions of the invention with an inert material.Such diluents can include carbohydrates, especially, mannitol,α-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans andstarch. Certain inorganic salts may also be used as fillers, includingcalcium triphosphate, magnesium carbonate and sodium chloride. Somecommercially available diluents are Fast-Flo, Emdex, STA-Rx 1500,Emcompress and Avicell.

A variety of conventional thickeners are useful in creams, ointments,suppository and gel configurations of the pharmaceutical composition,such as, but not limited to, alginate, xanthan gum, or petrolatum, mayalso be employed in such configurations of the pharmaceuticalcomposition of the present invention. A permeation or penetrationenhancer, such as polyethylene glycol monolaurate, dimethyl sulfoxide,N-vinyl-2-pyrrolidone, N-(2-hydroxyethyl)-pyrrolidone, or3-hydroxy-N-methyl-2-pyrrolidone can also be employed. Useful techniquesfor producing hydrogel matrices are known. (E.g., Feijen, Biodegradablehydrogel matrices for the controlled release of pharmacologically activeagents, U.S. Pat. No. 4,925,677; Shah et al., BiodegradablepH/thermosensitive hydrogels for sustained delivery of biologicallyactive agents, WO 00/38651 A1). Such biodegradable gel matrices can beformed, for example, by crosslinking a proteinaceous component and apolysaccharide or mucopolysaccharide component, then loading with theinventive composition of matter to be delivered.

Liquid pharmaceutical compositions of the present invention that aresterile solutions or suspensions can be administered to a patient byinjection, for example, intramuscularly, intrathecally, epidurally,intravascularly (e.g., intravenously or intrarterially),intraperitoneally or subcutaneously. (See, e.g., Goldenberg et al.,Suspensions for the sustained release of proteins, U.S. Pat. No.6,245,740 and WO 00/38652 A1). Sterile solutions can also beadministered by intravenous infusion. The inventive composition can beincluded in a sterile solid pharmaceutical composition, such as alyophilized powder, which can be dissolved or suspended at a convenienttime before administration to a patient using sterile water, saline,buffered saline or other appropriate sterile injectable medium.

Implantable sustained release formulations are also useful embodimentsof the inventive pharmaceutical compositions. For example, thepharmaceutically acceptable carrier, being a biodegradable matriximplanted within the body or under the skin of a human or non-humanvertebrate, can be a hydrogel similar to those described above.Alternatively, it may be formed from a poly-alpha-amino acid component.(Sidman, Biodegradable, implantable drug delivery device, and processfor preparing and using same, U.S. Pat. No. 4,351,337). Other techniquesfor making implants for delivery of drugs are also known and useful inaccordance with the present invention.

In powder forms, the pharmaceutically acceptable carrier is a finelydivided solid, which is in admixture with finely divided activeingredient(s), including the inventive composition. For example, in someembodiments, a powder form is useful when the pharmaceutical compositionis configured as an inhalant. (See, e.g., Zeng et al., Method ofpreparing dry powder inhalation compositions, WO 2004/017918; Trunk etal., Salts of the CGRP antagonist BIBN4096 and inhalable powderedmedicaments containing them, U.S. Pat. No. 6,900,317).

One can dilute or increase the volume of the compound of the inventionwith an inert material. These diluents could include carbohydrates,especially mannitol, α-lactose, anhydrous lactose, cellulose, sucrose,modified dextrans and starch. Certain inorganic salts can also be usedas fillers including calcium triphosphate, magnesium carbonate andsodium chloride. Some commercially available diluents are Fast-Flo™,Emdex™, STA-Rx™ 1500, Emcompress™ and Avicell™.

Disintegrants can be included in the formulation of the pharmaceuticalcomposition into a solid dosage form. Materials used as disintegrantsinclude but are not limited to starch including the commercialdisintegrant based on starch, Explotab™. Sodium starch glycolate,Amberlite™, sodium carboxymethylcellulose, ultramylopectin, sodiumalginate, gelatin, orange peel, acid carboxymethyl cellulose, naturalsponge and bentonite can all be used. Insoluble cationic exchange resinis another form of disintegrant. Powdered gums can be used asdisintegrants and as binders and these can include powdered gums such asagar, Karaya or tragacanth. Alginic acid and its sodium salt are alsouseful as disintegrants.

Binders can be used to hold the therapeutic agent together to form ahard tablet and include materials from natural products such as acacia,tragacanth, starch and gelatin. Others include methyl cellulose (MC),ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinylpyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both beused in alcoholic solutions to granulate the therapeutic.

An antifrictional agent can be included in the formulation of thetherapeutic to prevent sticking during the formulation process.Lubricants can be used as a layer between the therapeutic and the diewall, and these can include but are not limited to; stearic acidincluding its magnesium and calcium salts, polytetrafluoroethylene(PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricantscan also be used such as sodium lauryl sulfate, magnesium laurylsulfate, polyethylene glycol of various molecular weights, Carbowax 4000and 6000.

Glidants that might improve the flow properties of the drug duringformulation and to aid rearrangement during compression might be added.The glidants can include starch, talc, pyrogenic silica and hydratedsilicoaluminate.

To aid dissolution of the compound of this invention into the aqueousenvironment a surfactant might be added as a wetting agent. Surfactantscan include anionic detergents such as sodium lauryl sulfate, dioctylsodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergentsmight be used and could include benzalkonium chloride or benzethoniumchloride. The list of potential nonionic detergents that could beincluded in the formulation as surfactants are lauromacrogol 400,polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fattyacid ester, methyl cellulose and carboxymethyl cellulose. Thesesurfactants could be present in the formulation of the protein orderivative either alone or as a mixture in different ratios.

Oral dosage forms. Also useful are oral dosage forms of the inventivecompositionss. If necessary, the composition can be chemically modifiedso that oral delivery is efficacious. Generally, the chemicalmodification contemplated is the attachment of at least one moiety tothe molecule itself, where said moiety permits (a) inhibition ofproteolysis; and (b) uptake into the blood stream from the stomach orintestine. Also desired is the increase in overall stability of thecompound and increase in circulation time in the body. Moieties usefulas covalently attached half-life extending moieties in this inventioncan also be used for this purpose. Examples of such moieties include:PEG, copolymers of ethylene glycol and propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone andpolyproline. See, for example, Abuchowski and Davis (1981), SolublePolymer-Enzyme Adducts, Enzymes as Drugs (Hocenberg and Roberts, eds.),Wiley-Interscience, New York, N.Y., pp 367-83; Newmark, et al. (1982),J. Appl. Biochem. 4:185-9. Other polymers that could be used arepoly-1,3-dioxolane and poly-1,3,6-tioxocane. Preferred forpharmaceutical usage, as indicated above, are PEG moieties.

For oral delivery dosage forms, it is also possible to use a salt of amodified aliphatic amino acid, such as sodiumN-(8-[2-hydroxybenzoyl]amino)caprylate (SNAC), as a carrier to enhanceabsorption of the therapeutic compounds of this invention. The clinicalefficacy of a heparin formulation using SNAC has been demonstrated in aPhase II trial conducted by Emisphere Technologies. See U.S. Pat. No.5,792,451, “Oral drug delivery composition and methods.”

In one embodiment, the pharmaceutically acceptable carrier can be aliquid and the pharmaceutical composition is prepared in the form of asolution, suspension, emulsion, syrup, elixir or pressurizedcomposition. The active ingredient(s) (e.g., the inventive compositionof matter) can be dissolved, diluted or suspended in a pharmaceuticallyacceptable liquid carrier such as water, an organic solvent, a mixtureof both, or pharmaceutically acceptable oils or fats. The liquid carriercan contain other suitable pharmaceutical additives such as detergentsand/or solubilizers (e.g., Tween 80, Polysorbate 80), emulsifiers,buffers at appropriate pH (e.g., Tris-HCl, acetate, phosphate),adjuvants, anti-oxidants (e.g., ascorbic acid, sodium metabisulfite),preservatives (e.g., Thimersol, benzyl alcohol), sweeteners, flavoringagents, suspending agents, thickening agents, bulking substances (e.g.,lactose, mannitol), colors, viscosity regulators, stabilizers,electrolytes, osmolutes or osmo-regulators. Additives can also beincluded in the formulation to enhance uptake of the inventivecomposition. Additives potentially having this property are for instancethe fatty acids oleic acid, linoleic acid and linolenic acid.

Useful are oral solid dosage forms, which are described generally inRemington's Pharmaceutical Sciences (1990), supra, in Chapter 89, whichis hereby incorporated by reference in its entirety. Solid dosage formsinclude tablets, capsules, pills, troches or lozenges, cachets orpellets. Also, liposomal or proteinoid encapsulation can be used toformulate the present compositions (as, for example, proteinoidmicrospheres reported in U.S. Pat. No. 4,925,673). Liposomalencapsulation can be used and the liposomes can be derivatized withvarious polymers (e.g., U.S. Pat. No. 5,013,556). A description ofpossible solid dosage forms for the therapeutic is given in Marshall,K., Modern Pharmaceutics (1979), edited by G. S. Banker and C. T.Rhodes, in Chapter 10, which is hereby incorporated by reference in itsentirety. In general, the formulation will include the inventivecompound, and inert ingredients that allow for protection against thestomach environment, and release of the biologically active material inthe intestine.

The composition of this invention can be included in the formulation asfine multiparticulates in the form of granules or pellets of particlesize about 1 mm. The formulation of the material for capsuleadministration could also be as a powder, lightly compressed plugs oreven as tablets. The therapeutic could be prepared by compression.

Colorants and flavoring agents can all be included. For example, theprotein (or derivative) can be formulated (such as by liposome ormicrosphere encapsulation) and then further contained within an edibleproduct, such as a refrigerated beverage containing colorants andflavoring agents.

In tablet form, the active ingredient(s) are mixed with apharmaceutically acceptable carrier having the necessary compressionproperties in suitable proportions and compacted in the shape and sizedesired.

The powders and tablets preferably contain up to 99% of the activeingredient(s). Suitable solid carriers include, for example, calciumphosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch,gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ionexchange resins.

Controlled release formulation can be desirable. The composition of thisinvention could be incorporated into an inert matrix that permitsrelease by either diffusion or leaching mechanisms e.g., gums. Slowlydegenerating matrices can also be incorporated into the formulation,e.g., alginates, polysaccharides. Another form of a controlled releaseof the compositions of this invention is by a method based on the Oros™therapeutic system (Alza Corp.), i.e., the drug is enclosed in asemipermeable membrane which allows water to enter and push drug outthrough a single small opening due to osmotic effects. Some entericcoatings also have a delayed release effect.

Other coatings can be used for the formulation. These include a varietyof sugars that could be applied in a coating pan. The therapeutic agentcould also be given in a film-coated tablet and the materials used inthis instance are divided into 2 groups. The first are the nonentericmaterials and include methylcellulose, ethyl cellulose, hydroxyethylcellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose,hydroxypropyl-methyl cellulose, sodium carboxymethyl cellulose,providone and the polyethylene glycols. The second group consists of theenteric materials that are commonly esters of phthalic acid.

A mix of materials might be used to provide the optimum film coating.Film coating can be carried out in a pan coater or in a fluidized bed orby compression coating.

Pulmonary delivery forms. Pulmonary delivery of the inventivecompositions is also useful. The protein (or derivative) is delivered tothe lungs of a mammal while inhaling and traverses across the lungepithelial lining to the blood stream. (Other reports of this includeAdjei et al., Pharma. Res. (1990) 7: 565-9; Adjei et al. (1990),Internatl. J. Pharmaceutics 63: 135-44 (leuprolide acetate); Braquet etal. (1989), J. Cardiovasc. Pharmacol. 13 (suppl. 5): s. 143-146(endothelin-1); Hubbard et al. (1989), Annals Int. Med. 3: 206-12(α1-antitrypsin); Smith et al. (1989), J. Clin. Invest. 84: 1145-6(α1-proteinase); Oswein et al. (March 1990), “Aerosolization ofProteins,” Proc. Symp. Resp. Drug Delivery II, Keystone, Colo.(recombinant human growth hormone); Debs et al. (1988), J. Immunol. 140:3482-8 (interferon-γ and tumor necrosis factor α) and Platz et al., U.S.Pat. No. 5,284,656 (granulocyte colony stimulating factor). Useful inthe practice of this invention are a wide range of mechanical devicesdesigned for pulmonary delivery of therapeutic products, including butnot limited to nebulizers, metered dose inhalers, and powder inhalers,all of which are familiar to those skilled in the art. Some specificexamples of commercially available devices suitable for the practice ofthis invention are the Ultravent nebulizer, manufactured byMallinckrodt, Inc., St. Louis, Mo.; the Acorn II nebulizer, manufacturedby Marquest Medical Products, Englewood, Colo.; the Ventolin metereddose inhaler, manufactured by Glaxo Inc., Research Triangle Park, NorthCarolina; and the Spinhaler powder inhaler, manufactured by FisonsCorp., Bedford, Mass. (See, e.g., Helgesson et al., Inhalation device,U.S. Pat. No. 6,892,728; McDerment et al., Dry powder inhaler, WO02/11801 A1; Ohki et al., Inhalant medicator, U.S. Pat. No. 6,273,086).

All such devices require the use of formulations suitable for thedispensing of the inventive compound. Typically, each formulation isspecific to the type of device employed and can involve the use of anappropriate propellant material, in addition to diluents, adjuvantsand/or carriers useful in therapy.

The inventive compound should most advantageously be prepared inparticulate form with an average particle size of less than 10 μm (ormicrons), most preferably 0.5 to 5 μm, for most effective delivery tothe distal lung.

Pharmaceutically acceptable carriers include carbohydrates such astrehalose, mannitol, xylitol, sucrose, lactose, and sorbitol. Otheringredients for use in formulations can include DPPC, DOPE, DSPC andDOPC. Natural or synthetic surfactants can be used. PEG can be used(even apart from its use in derivatizing the protein or analog).Dextrans, such as cyclodextran, can be used. Bile salts and otherrelated enhancers can be used. Cellulose and cellulose derivatives canbe used. Amino acids can be used, such as use in a buffer formulation.

Also, the use of liposomes, microcapsules or microspheres, inclusioncomplexes, or other types of carriers is contemplated.

Formulations suitable for use with a nebulizer, either jet orultrasonic, will typically comprise the inventive compound dissolved inwater at a concentration of about 0.1 to 25 mg of biologically activeprotein per mL of solution. The formulation can also include a bufferand a simple sugar (e.g., for protein stabilization and regulation ofosmotic pressure). The nebulizer formulation can also contain asurfactant, to reduce or prevent surface induced aggregation of theprotein caused by atomization of the solution in forming the aerosol.

Formulations for use with a metered-dose inhaler device will generallycomprise a finely divided powder containing the inventive compoundsuspended in a propellant with the aid of a surfactant. The propellantcan be any conventional material employed for this purpose, such as achlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or ahydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, orcombinations thereof. Suitable surfactants include sorbitan trioleateand soya lecithin. Oleic acid can also be useful as a surfactant. (See,e.g., Bäckström et al., Aerosol drug formulations containinghydrofluoroalkanes and alkyl saccharides, U.S. Pat. No. 6,932,962).

Formulations for dispensing from a powder inhaler device will comprise afinely divided dry powder containing the inventive compound and can alsoinclude a bulking agent, such as lactose, sorbitol, sucrose, mannitol,trehalose, or xylitol in amounts which facilitate dispersal of thepowder from the device, e.g., 50 to 90% by weight of the formulation.

Nasal delivery forms. In accordance with the present invention,intranasal delivery of the inventive composition of matter and/orpharmaceutical compositions is also useful, which allows passage thereofto the blood stream directly after administration to the inside of thenose, without the necessity for deposition of the product in the lung.Formulations suitable for intransal administration include those withdextran or cyclodextran, and intranasal delivery devices are known.(See, e.g, Freezer, Inhaler, U.S. Pat. No. 4,083,368).

Transdermal and transmucosal (e.g., Buccal) delivery forms). In someembodiments, the inventive composition is configured as a part of apharmaceutically acceptable transdermal or transmucosal patch or atroche. Transdermal patch drug delivery systems, for example, matrixtype transdermal patches, are known and useful for practicing someembodiments of the present pharmaceutical compositions. (E.g., Chien etal., Transdermal estrogen/progestin dosage unit, system and process,U.S. Pat. Nos. 4,906,169 and 5,023,084; Cleary et al., Diffusion matrixfor transdermal drug administration and transdermal drug deliverydevices including same, U.S. Pat. No. 4,911,916; Teillaud et al.,EVA-based transdermal matrix system for the administration of anestrogen and/or a progestogen, U.S. Pat. No. 5,605,702; Venkateshwaranet al., Transdermal drug delivery matrix for coadministering estradioland another steroid, U.S. Pat. No. 5,783,208; Ebert et al., Methods forproviding testosterone and optionally estrogen replacement therapy towomen, U.S. Pat. No. 5,460,820). A variety of pharmaceuticallyacceptable systems for transmucosal delivery of therapeutic agents arealso known in the art and are compatible with the practice of thepresent invention. (E.g., Heiber et al., Transmucosal delivery ofmacromolecular drugs, U.S. Pat. Nos. 5,346,701 and 5,516,523;Longenecker et al., Transmembrane formulations for drug administration,U.S. Pat. No. 4,994,439).

Buccal delivery of the inventive compositions is also useful. Buccaldelivery formulations are known in the art for use with peptides. Forexample, known tablet or patch systems configured for drug deliverythrough the oral mucosa (e.g., sublingual mucosa), include someembodiments that comprise an inner layer containing the drug, apermeation enhancer, such as a bile salt or fusidate, and a hydrophilicpolymer, such as hydroxypropyl cellulose, hydroxypropyl methylcellulose,hydroxyethyl cellulose, dextran, pectin, polyvinyl pyrrolidone, starch,gelatin, or any number of other polymers known to be useful for thispurpose. This inner layer can have one surface adapted to contact andadhere to the moist mucosal tissue of the oral cavity and can have anopposing surface adhering to an overlying non-adhesive inert layer.Optionally, such a transmucosal delivery system can be in the form of abilayer tablet, in which the inner layer also contains additionalbinding agents, flavoring agents, or fillers. Some useful systems employa non-ionic detergent along with a permeation enhancer. Transmucosaldelivery devices may be in free form, such as a cream, gel, or ointment,or may comprise a determinate form such as a tablet, patch or troche.For example, delivery of the inventive composition can be via atransmucosal delivery system comprising a laminated composite of, forexample, an adhesive layer, a backing layer, a permeable membranedefining a reservoir containing the inventive composition, a peel sealdisc underlying the membrane, one or more heat seals, and a removablerelease liner. (E.g., Ebert et al., Transdermal delivery system withadhesive overlay and peel seal disc, U.S. Pat. No. 5,662,925; Chang etal., Device for administering an active agent to the skin or mucosa,U.S. Pat. Nos. 4,849,224 and 4,983,395). These examples are merelyillustrative of available transmucosal drug delivery technology and arenot limiting of the present invention.

Dosages. The dosage regimen involved in a method for treating theabove-described conditions will be determined by the attendingphysician, considering various factors which modify the action of drugs,e.g. the age, condition, body weight, sex and diet of the patient, theseverity of any infection, time of administration and other clinicalfactors. Generally, the daily regimen should be in the range of 0.1-1000micrograms of the inventive compound per kilogram of body weight,preferably 0.1-150 micrograms per kilogram.

WORKING EXAMPLES

The compositions described above can be prepared as described below.These examples are not to be construed in any way as limiting the scopeof the present invention.

Example 1 Fc-L10-ShK[1-35] Mammalian Expression

Fc-L10-ShK[1-35], also referred to as “Fc-2xL-ShK[1-35]”, an inhibitorof Kv1.3. A DNA sequence coding for the Fc region of human IgG1 fusedin-frame to a linker sequence and a monomer of the Kv1.3 inhibitorpeptide ShK[1-35] was constructed as described below. Methods forexpressing and purifying the peptibody from mammalian cells (HEK 293 andChinese Hamster Ovary cells) are disclosed herein.

The expression vector pcDNA3.1(+)CMVi (FIG. 13A) was constructed byreplacing the CMV promoter between MluI and HindIII in pcDNA3.1(+) withthe CMV promoter plus intron (Invitrogen). The expression vectorpcDNA3.1(+)CMVi-hFc-ActivinRIIB (FIG. 13B) was generated by cloning aHindIII-NotI digested PCR product containing a 5′ Kozak sequence, asignal peptide and the human Fc-linker—ActivinRIIB fusion protein withthe large fragment of HindIII-NotI digested pcDNA3.1(+)CMVi. Thenucleotide and amino acid sequence of the human IgG1 Fc region inpcDNA3.1(+)CMVi-hFc-ActivinRIIB is shown in FIG. 3. This vector also hasa GGGGSGGGGS (“L10”; SEQ ID NO:79) linker split by a BamHI site thusenabling with the oligo below formation of the 10 amino acid linkerbetween Fc and the ShK[1-35] peptide (see FIG. 14) for the finalFc-L10-ShK[1-35] nucleotide and amino acid sequence (FIG. 14 and SEQ IDNO: 77 and SEQ ID NO:78).

The Fc-L10-ShK[1-35] expression vector was constructing using PCRstategies to generate the full length ShK gene linked to a four glycineand one serine amino acid linker (lower case letters here indicatelinker sequence of L-form amino acid residues) with two stop codons andflanked by BamHI and NotI restriction sites as shown below.

BamHIGGATCCGGAGGAGGAGGAAGCCGCAGCTGCATCGACACCATCCCCAAGAGCCGCTGCACCGCCTTCCAG//SEQ ID NO: 657      g  g  g  g  s  R  S  C  I  D  T  I  P  K  S  R  C  T  A  F  Q//SEQ ID NO: 658TGCAAGCACAGCATGAAGTACCGCCTGAGCTTCTGCCGCAAGACCTGCGGCACCTGCTAATGAGCGGCCGCC  K  H  S  M  K  Y  R  L  S  F  C  R  K  T  C  G  T  C          NotI

Two oligos with the sequence as depicted below were used in a PCRreaction with Herculase™ polymerase (Stratagene) at 94° C.-30 sec, 50°C.-30 sec, and 72° C.-1 min for 30 cycles.

cat gga tcc gga gga gga gga agc //SEQ ID NO: 659cgc agc tgc atc gac acc atc ccc aag agc cgc tgc acc gcc ttc cagtgc aag cac cat gcg gcc gct cat tag cag gtg //SEQ ID NO: 660ccg cag gtc ttg cgg cag aag ctc agg cgg tac ttc atg ctg tgc ttgcac tgg aag g

The resulting PCR products were resolved as the 150 bp bands on a onepercent agarose gel. The 150 bp PCR product was digested with BamHI andNotI (Roche) restriction enzymes and agarose gel purified by GelPurification Kit (Qiagen). At the same time, thepcDNA3.1(+)CMVi-hFc-ActivinRIIB vector (FIG. 13B ) was digested withBamHI and NotI restriction enzymes and the large fragment was purifiedby Gel Purification Kit. The gel purified PCR fragment was ligated tothe purified large fragment and transformed into XL-1blue bacteria(Stratagene). DNAs from transformed bacterial colonies were isolated anddigested with BamHI and NotI restriction enzyme digestion and resolvedon a one percent agarose gel. DNAs resulting in an expected pattern weresubmitted for sequencing. Although, analysis of several sequences ofclones yielded a 100% percent match with the above sequence, only oneclone was selected for large scaled plasmid purification. The DNA fromFc-2xL-ShK in pcDNA3.1(+)CMVi clone was resequenced to confirm the Fcand linker regions and the sequence was 100% identical to the predictedcoding sequence, which is shown in FIG. 14.

HEK-293 cells used in transient transfection expression ofFc-2xL-ShK[1-35] in pcDNA3.1(+)CMVi protein were cultured in growthmedium containing DMEM High Glucose (Gibco), 10% fetal bovine serum (FBSfrom Gibco) and 1× non-essential amino acid (NEAA from Gibco). 5.6 ug ofFc-2xL-ShK[1-35] in pcDNA3.1(+)CMVi plasmid that had beenphenol/chloroform extracted was transfected into HEK-293 cells usingFugene 6 (Roche). The cells recovered for 24 hours, and then placed inDMEM High Glucose and 1×NEAA medium for 48 hours. The conditioned mediumwas concentrated 50× by running 30 ml through Centriprep YM-10 filter(Amicon) and further concentrated by a Centricon YM-10 (Amicon) filter.Various amounts of concentrated medium were mixed with an in-house 4×Loading Buffer (without B-mercaptoethanol) and electrophoresed on aNovex 4-20% tris-glycine gel using a Novex Xcell II apparatus at 101V/46mA for 2 hours in a 5× Tank buffer solution (0.123 Tris Base, 0.96MGlycine) along with 10 ul of BenchMark Pre-Stained Protein ladder(Invitrogen). The gel was then soaked in Electroblot buffer (35 mM Trisbase, 20% methanol, 192 mM glycine) for 30 minutes. A PVDF membrane fromNovex (Cat. No. LC2002, 0.2 um pores size) was soaked in methanol for 30seconds to activate the PVDF, rinsed with deionized water, and soaked inElectroblot buffer. The pre-soaked gel was blotted to the PVDF membraneusing the XCell II Blot module according to the manufacturerinstructions (Novex) at 40 mA for 2 hours. Then, the blot was firstsoaked in a 5% milk (Carnation) in Tris buffered saline solution pH 7.5(TBS) for 1 hour at room temperature and incubated with 1:500 dilutionin TBS with 0.1% Tween-20 (TBST Sigma) and 1% milk buffer of theHRP-conjugated murine anti-human Fc antibody (Zymed Laboratores Cat. no.05-3320) for two hours shaking at room temperature. The blot was thenwashed three times in TBST for 15 minutes per wash at room temperature.The primary antibody was detected using Amersham Pharmacia Biotech's ECLwestern blotting detection reagents according to manufacturer'sinstructions. Upon ECL detection, the western blot analysis displayedthe expected size of 66 kDa under non-reducing gel conditions (FIG.24A).

AM1 CHOd-(Amgen Proprietary) cells used in the stable expression ofFc-L10-ShK[1-35] protein were cultured in AM1 CHOd-growth mediumcontaining DMEM High Glucose, 10% fetal bovine serum, 1×hypoxantine/thymidine (HT from Gibco) and 1×NEAA. 6.5 ug ofpcDNA3.1(+)CMVi-Fc-ShK plasmid was also transfected into AM1 CHOd-cellsusing Fugene 6. The following day, the transfected cells were platedinto twenty 15 cm dishes and selected using DMEM high glucose, 10% FBS,1×HT, 1×NEAA and Geneticin (800ug/ml G418 from Gibco) for thirteen days.Forty-eight surviving colonies were picked into two 24-well plates. Theplates were allowed to grow up for a week and then replicated forfreezing. One set of each plate was transferred to AM1 CHOd-growthmedium without 10% FBS for 48 hours and the conditioned media wereharvested. Western Blot analysis similar to the transient Western blotanalysis with detection by the same anti-human Fc antibody was used toscreen 15 ul of conditioned medium for expressing stable CHO clones. Ofthe 48 stable clones, more than 50% gave ShK expression at the expectedsize of 66 kDa. The BB6, BD5 and BD6 clones were selected with BD5 andBD6 as a backup to the primary clone BB6 (FIG. 24B).

The BB6 clone was scaled up into ten roller bottles (Corning) using AM1CHOd-growth medium and grown to confluency as judged under themicroscope. Then, the medium was exchanged with a serum-free mediumcontaining to 50% DMEM high glucose and 50% Ham's F12 (Gibco) with 1×HTand 1×NEAA and let incubate for one week. The conditioned medium washarvested at the one-week incubation time, filtered through 0.45 μmfilter (Corning) and frozen. Fresh serum-free medium was added andincubated for an additional week. The conditioned serum-free medium washarvested like the first time and frozen.

Approximately 4 L of conditioned medium was thawed in a water bath atroom temperature. The medium was concentrated to about 450 ml using aSatorius Sartocon Polysulfon 10 tangential flow ultra-filtrationcassette (0.1 m²) at room temperature. The retentate was then filteredthrough a 0.22 μm cellulose acetate filter with a pre-filter. Theretentate was then loaded on to a 5 ml Amersham HiTrap Protein A columnat 5 ml/min 7° C., and the column was washed with several column volumesof Dulbecco's phosphate buffered saline without divalent cations (PBS)and sample was eluted with a step to 100 mM glycine pH 3.0. The proteinA elution pool (approximately 9 ml) was diluted to 50 ml with water andloaded on to a 5 ml Amersham HiTrap SP-HP column in S-Buffer A (20 mMNaH₂PO₄, pH 7.0) at 5 ml/min and 7° C. The column was then washed withseveral column volumes S-Buffer A, and then developed using a lineargradient from 25% to 75% S-Buffer B (20 mM NaH₂PO₄, 1 M NaCl, pH 7.0) at5 ml/min followed by a step to 100% S-Buffer B at 7° C. Fractions werethen analyzed using a Coomassie brilliant blue stained tris-glycine4-20% SDS-PAGE, and the fractions containing the desired product werepooled based on these data. The pooled material was then concentrated toabout 3.4 ml using a Pall Life Sciences Macrosep 10K Omega centrifugalultra-filtration device and then filtered though a Costar 0.22 μmcellulose acetate syringe filter.

A spectral scan was then conducted on 10 μl of the filtered materialdiluted in 700 μl PBS using a Hewlett Packard 8453 spectrophotometer(FIG. 26A). The concentration of the filtered material was determined tobe 5.4 mg/ml using a calculated molecular mass of 32,420 g/mol andextinction coefficient of 47,900 M⁻¹ cm⁻¹. The purity of the filteredmaterial was then assessed using a Coomassie brilliant blue stainedtris-glycine 4-20% SDS-PAGE (FIG. 26B). The endotoxin level was thendetermined using a Charles River Laboratories Endosafe-PTS system(0.05-5 EU/ml sensitivity) using a 108-fold dilution of the sample inPBS yielding a result of <1 EU/mg protein. The macromolecular state ofthe product was then determined using size exclusion chromatography on20 μg of the product injected on to a Phenomenex BioSep SEC 3000 column(7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observingthe absorbance at 280 nm (FIG. 26C). The product was then subject tomass spectral analysis by diluting 1 μl of the sample into 10 μl ofsinapinic acid (10 mg per ml in 0.05% trifluroacetic acid, 50%acetonitrile). The resultant solution (1 μl) was spotted onto a MALDIsample plate. The sample was allowed to dry before being analyzed usinga Voyager DE-RP time-of-flight mass spectrometer equipped with anitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode wasused, with an accelerating voltage of 25 kV. Each spectrum was producedby accumulating data from ˜200 laser shots. External mass calibrationwas accomplished using purified proteins of known molecular masses (FIG.26D) and confirmed (within experimental error) the integrity of thepurified peptibody. The product was then stored at −80° C.

Purified Fc-L10-ShK[1-35] potently blocked human Kv1.3 (FIG. 30A andFIG. 30B) as determined by electrophysiology (see Example 36). Thepurified Fc-L10-ShK[1-35] molecule also blocked T cell proliferation(FIG. 36A and FIG. 36B) and production of the cytokines IL-2 (FIG. 35Aand FIG. 37A) and IFN-g (FIG. 35B and FIG. 37B).

Example 2 Fc-L-ShK[2-35] Mammalian Expression

A DNA sequence coding for the Fc region of human IgG1 fused in-frame toa monomer of the Kv1.3 inhibitor peptide ShK[2-35] was constructed usingstandard PCR technology. The ShK[2-35] and the 5, 10, or 25 amino acidlinker portion of the molecule were generated in a PCR reaction usingthe original Fc-2xL-ShK[1-35] in pcDNA3.1(+)CMVi as a template (Example1, FIG. 14). All ShK constructs should have the following amino acidsequence of

SCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC (SEQ ID NO: 92)with the first amino acid of the wild-type sequence deleted.

The sequences of the primers used to generate Fc-L5-ShK[2-35], alsoreferred to as “Fc-1xL-ShK[2-35]”, are shown below:

cat gga tcc agc tgc atc gac acc //SEQ ID NO: 661 atc;cat gcg gcc gct cat tag c; //SEQ ID NO: 662

The sequences of the primers used to generate Fc-L10-ShK[2-35], alsoreferred to as “Fc-2xL-ShK[2-35]” are shown below:

cat gga tcc gga gga gga gga agc //SEQ ID NO: 663 agc tgc a;cat gcg gcc gct cat tag cag gtg c; //SEQ ID NO: 664

The sequences of the primers used to generate Fc-L25-ShK[2-35], alsoreferred to as “Fc-5xL-ShK[2-35]”, are shown below:

cat gga tcc ggg ggt ggg ggt tct //SEQ ID NO: 665ggg ggt ggg ggt tct gga gga gga gga agc gga gga gga gga agc agc tgc a;cat gcg gcc gct cat tag cag gtg c; //SEQ ID NO: 666

The PCR products were digested with BamHI and NotI (Roche) restrictionenzymes and agarose gel purified by Gel Purification Kit. At the sametime, the pcDNA3.1(+)CMVi-hFc-ActivinRIIB vector was digested with BamHIand NotI restriction enzymes and the large fragment was purified by GelPurification Kit. Each purified PCR product was ligated to the largefragment and transformed into XL-1blue bacteria. DNAs from transformedbacterial colonies were isolated and subjected to BamHI and NotIrestriction enzyme digestions and resolved on a one percent agarose gel.DNAs resulting in an expected pattern were submitted for sequencing.Although, analysis of several sequences of clones yielded a 100% percentmatch with the above sequence, only one clone was selected for largescaled plasmid purification. The DNA from this clone was resequenced toconfirm the Fc and linker regions and the sequence was 100% identical tothe expected sequence.

Plasmids containing the Fc-1xL-Shk[2-35], Fc-2xL-Shk[2-35] andFc-5xL-Shk[2-35] inserts in pcDNA3.1 (+)CMVi vector were digested withXba1 and Xho1 (Roche) restriction enzymes and gel purified. The insertswere individually ligated into Not1 and SaII (Roche) digested pDSRα-22(Amgen Proprietary) expression vector. Integrity of the resultingconstructs were confirmed by DNA sequencing. The final plasmid DNAexpression vector constructs were pDSRα-22-Fc-1xL-Shk[2-35],pDSRα-22-Fc-2xL-Shk[2-35] (FIG. 13C and FIG. 15) andpDSRα-22-Fc-5xL-Shk[2-35] (FIG. 16) and contained 5, 10 and 25 aminoacid linkers, respectively.

Twenty-four hours prior to transfection, 1.2e7 AM-1/D CHOd-(AmgenProprietary) cells were plated into a T-175 cm sterile tissue cultureflask, to allow 70-80% confluency on the day of transfection. The cellshad been maintained in the AM-1/D CHOd-culture medium containing DMEMHigh Glucose, 5% FBS, 1× Glutamine Pen/Strep (Gibco), 1×HT, 1×NEAA's and1× sodium pyruvate (Gibco). The following day, eighteen micrograms ofeach of the linearized pDSRα22:Fc-1xL-ShK[2-35],pDSRα22:Fc-2xL-ShK[2-35] and pDSRα22:Fc-5xL-ShK[2-35] (RDS's #20050037685, 20050053709, 20050073295) plasmids were mixed with 72 μg oflinearized Selexis MAR plasmid and pPAGO1 (RDS 20042009896) and dilutedinto 6 ml of OptiMEM in a 50 ml conical tube and incubate for fiveminutes. LF2000 (210 μl) was added to 6 ml of OptiMEM and incubated forfive minutes. The diluted DNA and LF2000 were mixed together andincubated for 20 minutes at room temperature. In the meantime, the cellswere washed one time with PBS and then 30 ml OptiMEM without antibioticswere added to the cells. tThe OptiMEM was aspirated off, and the cellswere incubated with 12 ml of DNA/LF2000 mixture for 6 hours or overnightin the 37° C. incubator with shaking. Twenty-four hours posttransfection, the cells were split 1:5 into AM-1/D CHOd-culture mediumand at differing dilutions for colony selection. Seventy-two hours posttransfection, the cell medium was replaced with DHFR selection mediumcontaining 10% Dialyzed FBS (Gibco) in DMEM High Glucose, plus 1×Glutamine Pen/Strep, 1×NEAA's and 1×Na Pyr to allow expression andsecretion of protein into the cell medium. The selection medium waschanged two times a week until the colonies are big enough to pick. ThepDSRa22 expression vector contains a DHFR expression cassette, whichallows transfected cells to grow in the absence of hypoxanthine andthymidine. The five T-175 pools of the resulting colonies were scaled upinto roller bottles and cultured under serum free conditions. Theconditioned media were harvested and replaced at one-week intervals. Theresulting 3 liters of conditioned medium was filtered through a 0.45 umcellulose acetate filter (Corning, Acton, Mass.) and transferred toProtein Chemistry for purification. As a backup, twelve colonies wereselected from the 10 cm plates after 10-14 days on DHFR selection mediumand expression levels evaluated by western blot using HRP conjugatedanti human IgGFc as a probe. The three best clones expressing thehighest level of each of the different linker length Fc-L-ShK[2-35]fusion proteins were expanded and frozen for future use.

Purification of Fc-L10-ShK(2-35). Approximately 1 L of conditionedmedium was thawed in a water bath at room temperature. The medium wasloaded on to a 5 ml Amersham HiTrap Protein A column at 5 ml/min 7° C.,and the column was washed with several column volumes of Dulbecco'sphosphate buffered saline without divalent cations (PBS) and sample waseluted with a step to 100 mM glycine pH 3.0. The protein A elution pool(approximately 8.5 ml) combined with 71 μl 3 M sodium acetate and thendiluted to 50 ml with water. The diluted material was then loaded on toa 5 ml Amersham HiTrap SP-HP column in S-Buffer A (20 mM NaH₂PO₄, pH7.0) at 5 ml/min 7° C. The column was then washed with several columnvolumes S-Buffer A, and then developed using a linear gradient from 0%to 75% S-Buffer B (20 mM NaH₂PO₄, 1 M NaCl, pH 7.0) at 5 ml/min followedby a step to 100% S-Buffer B at 7° C. Fractions were then analyzed usinga Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE, and thefractions containing the desired product were pooled based on thesedata. The pooled material was then filtered through a 0.22 μm celluloseacetate filter and concentrated to about 3.9 ml using a Pall LifeSciences Macrosep 10K Omega centrifugal ultra-filtration device. Theconcentrated material was then filtered though a Pall Life SciencesAcrodisc with a 0.22 μm, 25 mm Mustang E membrane at 2 ml/min roomtemperature. A spectral scan was then conducted on 10 μl of the filteredmaterial diluted in 700 μl PBS using a Hewlett Packard 8453spectrophotometer (FIG. 27E). The concentration of the filtered materialwas determined to be 2.76 mg/ml using a calculated molecular mass of30,008 g/mol and extinction coefficient of 36,900 M⁻¹ cm⁻¹. Sincematerial was found in the permeate, repeated concentration step on thepermeate using a new Macrosep cartridge. The new batch of concentratedmaterial was then filtered though a Pall Life Sciences Acrodisc with a0.22 μm, 25 mm Mustang E membrane at 2 ml/min room temperature. Bothlots of concentrated material were combined into one pool.

A spectral scan was then conducted on 10 μL of the combined pool dilutedin 700 μl PBS using a Hewlett Packard 8453 spectrophotometer. Theconcentration of the filtered material was determined to be 3.33 mg/mlusing a calculated molecular mass of 30,008 g/mol and extinctioncoefficient of 36,900 M⁻¹ cm⁻¹. The purity of the filtered material wasthen assessed using a Coomassie brilliant blue stained tris-glycine4-20% SDS-PAGE (FIG. 27A). The endotoxin level was then determined usinga Charles River Laboratories Endosafe-PTS system (0.05-5 EU/mlsensitivity) using a 67-fold dilution of the sample in PBS yielding aresult of <1 EU/mg protein. The macromolecular state of the product wasthen determined using size exclusion chromatography on 50 μg of theproduct injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm)in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observing theabsorbance at 280 nm (FIG. 27B). The product was then subject to massspectral analysis by diluting 1 μl of the sample into 10 μl of sinapinicacid (10 mg per ml in 0.05% trifluroacetic acid, 50% acetonitrile). Theresultant solution (1 μl) was spotted onto a MALDI sample plate. Thesample was allowed to dry before being analyzed using a Voyager DE-RPtime-of-flight mass spectrometer equipped with a nitrogen laser (337 nm,3 ns pulse). The positive ion/linear mode was used, with an acceleratingvoltage of 25 kV. Each spectrum was produced by accumulating data from˜200 laser shots. External mass calibration was accomplished usingpurified proteins of known molecular masses (FIG. 27F) and theexperiment confirmed the itegrity of the peptibody, within experimentalerror. The product was then stored at −80° C.

FIG. 31B shows that purified Fc-L10-ShK[2-35] potently blocks humanKv1.3 current (electrophysiology was done as described in Example 36).The purified Fc-L10-ShK[2-35] molecule also blocked IL-2 (FIG. 64A andFIG. 64B) and IFN-g (FIG. 65A and FIG. 65B) production in human wholeblood, as well as, upregulation of CD40L (FIG. 66A and FIG. 66B) andIL-2R (FIG. 67A and FIG. 67B) on T cells.

Purification of Fc-L5-ShK(2-35). Approximately 1 L of conditioned mediumwas loaded on to a 5 ml Amersham HiTrap Protein A column at 5 ml/min 7°C., and the column was washed with several column volumes of Dulbecco'sphosphate buffered saline without divalent cations (PBS) and sample waseluted with a step to 100 mM glycine pH 3.0. The protein A elution pool(approximately 9 ml) combined with 450 μl 1 M tris HCl pH 8.5 followedby 230 μl 2 M acetic acid and then diluted to 50 ml with water. The pHadjusted material was then filtered through a 0.22 μm cellulose acetatefilter and loaded on to a 5 ml Amersham HiTrap SP-HP column in S-BufferA (20 mM NaH₂PO₄, pH 7.0) at 5 ml/min 7° C. The column was then washedwith several column volumes S-Buffer A, and then developed using alinear gradient from 0% to 75% S-Buffer B (20 mM NaH₂PO₄, 1 M NaCl, pH7.0) at 5 ml/min followed by a step to 100% S-Buffer B at 7° C.Fractions were then analyzed using a Coomassie brilliant blue stainedtris-glycine 4-20% SDS-PAGE, and the fractions containing the desiredproduct were pooled based on these data. The pooled material was thenconcentrated to about 5.5 ml using a Pall Life Sciences Macrosep 10KOmega centrifugal ultra-filtration device. The concentrated material wasthen filtered though a Pall Life Sciences Acrodisc with a 0.22 μm, 25 mmMustang E membrane at 2 ml/min room temperature.

A spectral scan was then conducted on 10 μl of the combined pool dilutedin 700 μl PBS using a Hewlett Packard 8453 spectrophotometer (FIG. 27G).The concentration of the filtered material was determined to be 4.59mg/ml using a calculated molecular mass of 29,750 g/mol and extinctioncoefficient of 36,900 M⁻¹ cm⁻¹. The purity of the filtered material wasthen assessed using a Coomassie brilliant blue stained tris-glycine4-20% SDS-PAGE (FIG. 27C). The endotoxin level was then determined usinga Charles River Laboratories Endosafe-PTS system (0.05-5 EU/mlsensitivity) using a 92-fold dilution of the sample in Charles RiversEndotoxin Specific Buffer BG120 yielding a result of <1 EU/mg protein.The macromolecular state of the product was then determined using sizeexclusion chromatography on 50 μg of the product injected on to aPhenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mMNaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 27H).The product was then subject to mass spectral analysis by diluting 1 μlof the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05%trifluroacetic acid, 50% acetonitrile). The resultant solution (1 μl)was spotted onto a MALDI sample plate. The sample was allowed to drybefore being analyzed using a Voyager DE-RP time-of-flight massspectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). Thepositive ion/linear mode was used, with an accelerating voltage of 25kV. Each spectrum was produced by accumulating data from ˜200 lasershots. External mass calibration was accomplished using purifiedproteins of known molecular masses (FIG. 27I) and confirmed theintegrity of the peptibody, within experimental error. The product wasthen stored at −80° C.

FIG. 31C shows that purified Fc-L5-ShK[2-35] is highly active and blockshuman Kv1.3 as determined by whole cell patch clamp electrophysiology(see Example 36).

Purification of Fc-L25-ShK(2-35). Approximately 1 L of conditionedmedium was loaded on to a 5 ml Amersham HiTrap Protein A column at 5ml/min 7° C., and the column was washed with several column volumes ofDulbecco's phosphate buffered saline without divalent cations (PBS) andsample was eluted with a step to 100 mM glycine pH 3.0. The protein Aelution pool (approximately 9.5 ml) combined with 119 μl 3 M sodiumacetate and then diluted to 50 ml with water. The pH adjusted materialwas then loaded on to a 5 ml Amersham HiTrap SP-HP column in S-Buffer A(20 mM NaH₂PO₄, pH 7.0) at 5 ml/min 7° C. The column was then washedwith several column volumes S-Buffer A, and then developed using alinear gradient from 0% to 75% S-Buffer B (20 mM NaH₂PO₄, 1 M NaCl, pH7.0) at 5 ml/min followed by a step to 100% S-Buffer B at 7° C.Fractions containing the main peak from the chromatogram were pooled andfiltered through a 0.22 μm cellulose acetate filter.

A spectral scan was then conducted on 20 μl of the combined pool dilutedin 700 μl PBS using a Hewlett Packard 8453 spectrophotometer FIG. 27J.The concentration of the filtered material was determined to be 1.40mg/ml using a calculated molecular mass of 31,011 g/mol and extinctioncoefficient of 36,900 M⁻¹ cm⁻¹. The purity of the filtered material wasthen assessed using a Coomassie brilliant blue stained tris-glycine4-20% SDS-PAGE (FIG. 27D). The endotoxin level was then determined usinga Charles River Laboratories Endosafe-PTS system (0.05-5 EU/mlsensitivity) using a 28-fold dilution of the sample in Charles RiversEndotoxin Specific Buffer BG120 yielding a result of <1 EU/mg protein.The macromolecular state of the product was then determined using sizeexclusion chromatography on 50 μg of the product injected on to aPhenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mMNaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 27K).The product was then subject to mass spectral analysis by diluting 1 μlof the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05%trifluroacetic acid, 50% acetonitrile). The resultant solution (1 μl)was spotted onto a MALDI sample plate. The sample was allowed to drybefore being analyzed using a Voyager DE-RP time-of-flight massspectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). Thepositive ion/linear mode was used, with an accelerating voltage of 25kV. Each spectrum was produced by accumulating data from ˜200 lasershots. External mass calibration was accomplished using purifiedproteins of known molecular masses (FIG. 27L) and this confirmed theitegrity of the peptibody, within experimental error. The product wasthen stored at −80° C.

Purified Fc-L25-ShK[2-35] inhibited human Kv1.3 with an IC₅₀ of ˜150 pMby whole cell patch clamp electrophysiology on HEK293/Kv1.3 cells(Example 36).

Example 3 Fc-L-ShK[1-35] Bacterial Expression

Description of bacterial peptibody expression vectors and procedures forcloning and expression of peptibodies. The cloning vector used forbacterial expression (Examples 3-30) is based on pAMG21 (originallydescribed in U.S. Patent 2004/0044188). It has been modified in that thekanamycin resistance component has been replaced with ampicillinresistance by excising the DNA between the unique BstBI and NsiI sitesof the vector and replacing with an appropriately digested PCR fragmentbearing the beta-lactamase gene using PCR primers CCA ACA CAC TTC GAAAGA CGT TGA TCG GCA C (SEQ ID NO: 667) and CAC CCA ACA ATG CAT CCT TAAAAA AAT TAC GCC C (SEQ ID NO: 668) with pUC19 DNA as the template sourceof the beta-lactamase gene conferring resistance to ampicillin. The newversion is called pAMG21ampR.

Description of cloning vector pAMG21ampR-Fc-Pep used in examples 3 to30, excluding 15 and 16. FIG. 11A-C and FIG. 11D (schematic diagram)show the ds-DNA that has been added to the basic vector pAMG21ampR topermit the cloning of peptide fusions to the C-terminus of the Fc gene.The DNA has been introduced between the unique NdeI and BamHI sites inthe pAMG21ampR vector. This entire region of DNA is shown in FIG. 11A-C.The coding region for Fc extends from nt 5134 to 5817 and the proteinsequence appears below the DNA sequence. This is followed in frame by aglyX5 linker (nt's 5818-5832). A BsmBI site (GAGACG) spans nt's5834-5839. DNA cleavage occurs between nt's 5828 and 5829 on the upperDNA strand and between nt's 5832 and 5833 on the lower DNA strand.Digestion creates 4 bp cohesive termini as shown here. The BsmBI site isunderlined.

AGGTGG TGGTTGAGACG SEQ ID NO: 683 TCCACCACCA     ACTCTGC SEQ ID NO: 684

A second BsmBI site occurs at nt's 6643 through 6648; viz., CGTCTC. DNAcleavage occurs between nt's 6650 and 6651 on the upper strand andbetween 6654 and 6655 on the lower strand.

CGTCTCT TAAGGATCCG SEQ ID NO: 685 GCAGAGAATTC     CTAGGC SEQ ID NO: 686

Between the two BsmBI sites is a dispensable chloramphenicol resistancecassette constitutively expressing chloramphenicol acetyltransferase(cat gene). The cat protein sequence:

//SEQ ID NO: 1337 1 MEKKITGYTT VDISQWHRKE HFEAFQSVAQ CTYNQTVQLDITAFLKTVKK 51 NKHKFYPAFI HILARLMNAH PEFRMAMKDG ELVIWDSVHP CYTVFHEQTE 101TFSSLWSEYH DDFRQFLHIY SQDVACYGEN LAYFPKGFIE NMFFVSANPW 151VSFTSFDLNV ANMDNFFAPV FTMGKYYTQG DKVLMPLAIQ VHHAVCDGFH 201VGRMLNELQQ YCDEWQGGAis shown in FIG. 11A-C and extends from nt's 5954 to 6610. The peptideencoding duplexes in each example (except Examples 15 and 16) bearcohesive ends complementary to those presented by the vector.

Description of the cloning vector pAMG21ampR-Pep-Fc used in examples 15and 16. FIG. 12A-C, and the schematic diagram in FIG. 12D, shows theds-DNA sequence that has been added to the basic vector pAMG21ampR topermit the cloning of peptide fusions to the N-terminus of the Fc gene.The DNA has been introduced between the unique NdeI and BamHI sites inthe pAMG21ampR vector. The coding region for Fc extends from nt 5640 to6309 and the protein sequence appears below the DNA sequence. This ispreceded in frame by a glyX5 linker (nt's 5614-5628). A BsmBI site spansnt's 5138 to 5143; viz., GAGACG. The cutting occurs between nt's 5132and 5133 on the upper DNA strand and between 5136 and 5137 on the lowerDNA strand.

Digestion creates 4 bp cohesive termini as shown. The BsmBI site isunderlined.

AATAACA TATGCGAGACG SEQ ID NO: 687 TTATTGTATAC     GCTCTGCSEQ ID NO: 688

A second BsmBI site occurs at nt's 5607 through 5612; viz., CGTCTC.Cutting occurs between nt's 5613 and 5614 on the upper strand andbetween 5617 and 5618 on the lower strand.

CGTCTCA GGTGGTGGT SEQ ID NO: 689 GCAGAGTCCAC     CACCA

Between the BsmBI sites is a dispensable zeocin resistance cassetteconstitutively expressing the Shigella ble protein. The ble proteinsequence:

//SEQ ID NO: 1338 1 MAKLTSAVPV LTARDVAGAV EFWTDRLGFS RDFVEDDFAGVVRDDVTLFI 51 SAVQDQVVPD NTLAWVWVRG LDELYAEWSE VVSTNFRDAS GPAMTEIGEQ 101PWGREFALRD PAGNCVHFVA EEQDis shown extending from nt's 5217 to 5588 in FIG. 12A-C. The peptideencoding duplexes in Examples 15 and 16 bear cohesive ends complementaryto those presented by the vector.

Description of the cloning vector pAMG21ampR-Pep-Fc used in Examples 52and 53. FIG. 12E-F shows the ds-DNA sequence that has been added to thebasic vector pAMG21ampR to permit the cloning of peptide fusions to theN-terminus of the Fc gene in which the first two codons of the peptideare to be met-gly. The DNA has been introduced between the unique NdeIand BamHI sites in the pAMG21ampR vector. The coding region for Fcextends from nt 5632 to 6312 and the protein sequence appears below theDNA sequence. This is preceded in frame by a glyX5 linker (nt's5617-5631). A BsmBI site spans nt's 5141 to 5146; viz., GAGACG. Thecutting occurs between nt's 5135 and 5136 on the upper DNA strand andbetween 5139 and 5140 on the lower DNA strand.

Digestion creates 4 bp cohesive termini as shown. The BsmBI site isunderlined.

AATAACATAT GGGTCGAGACG SEQ ID NO: 1344 SEQ ID NO: 1343 TTATTGTATACCCA    GCTCTGC SEQ ID NO: 1345A second BsmBI site occurs at nt's 5607 through 5612; viz., CGTCTC.Cutting occurs between nt's 5613 and 5614 on the upper strand andbetween 5617 and 5618 on the lower strand.

CGTCTCA GGTGGTGGT SEQ ID NO: 1346 GCAGAGTCCAC     CACCABetween the BsmBI sites is a dispensable zeocin resistance cassetteconstitutively expressing the Shigella ble protein. The ble proteinsequence, as described above, is shown extending from nt's 5220 to 5591.The peptide encoding duplexes in Examples 52 and 53 herein below bearcohesive ends complementary to those presented by the vector.

For Examples 3 to 30 for which all are for bacterial expression, clonedpeptide sequences are all derived from the annealing of oligonucleotidesto create a DNA duplex that is directly ligated into the appropriatevector. Two oligos suffice for Example 20, four are required for allother examples. When the duplex is to be inserted at the N-terminus ofFc (see, Examples 15, 16, 52, and 53 herein) the design is as followswith the ordinal numbers matching the listing of oligos in each example:

When the duplex is to be inserted at the C-terminus of Fc (Examples 3,4, 5, 10, 11, 12, 13, and 30) the design is as follows:

All remaining examples have the duplex inserted at the C-terminus of Fcand utilize the following design.

No kinasing step is required for the phosphorylation of any of theoligos. A successful insertion of a duplex results in the replacement ofthe dispensable antibiotic resistance cassette (Zeocin resistance forpAMG21ampR-Pep-Fc and chloramphenicol resistance for pAMG21ampR-Fc-Pep).The resulting change in phenotype is useful for discriminatingrecombinant from nonrecombinant clones.

The following description gives the uniform method for carrying out thecloning of all 30 bacterially expressed recombinant proteins exemplifiedherein. Only the set of oligonucleotides and the vector are varied.These spefications are given below in each example.

An oligonucleotide duplex containing the coding region for a givenpeptide was formed by annealing the oligonucleotides listed in eachexample. Ten picomoles of each oligo was mixed in a final volume of 10μl containing 1× ligation buffer along with 0.3 μg of appropriate vectorthat had been previously digested with restriction endonuclease BsmBI.The mix was heated to 80° C. and allowed to cool at 0.1 degree/sec toroom temperature. To this was added 10 μl of 1× ligase buffer plus 400units of T4 DNA ligase. The sample was incubated at 14 C for 20 min.Ligase was inactivated by heating at 65° C. for 10 minutes. Next, 10units of restriction endonucleases BsmBI were added followed byincubation at 55 C for one hour to cleave any reformed parental vectormolecules. Fifty ul of chemically competent E. coli cells were added andheld at 2 C for 20 minutes followed by heat shock at 42 C for 5 second.The entire volume was spread onto Luria Agar plates supplemented withcarbenicillin at 200 ug/ml and incubated overnight at 37 C. Colonieswere tested for the loss of resistance to the replaceable antibioticresistance marker. A standard PCR test can be used to confirm theexpected size of the duplex insert. Plasmid preparations were obtainedand the recombinant insert was verified by DNA sequencing. Half litercultures of a sequence confirmed construct were grown in Terrific Broth,expression of the peptibody was induced by addition ofN-(3-oxo-hexanoyl)-homoserine lactone at 50 ng/ml and after 4-6 hours ofshaking at 37 C the cells were centrifuged and the cell paste stored at−20 C.

The following gives for each example the cloning vector and the set ofoligonucleotides used for constructing each fusion protein. Also shownis a DNA/protein map.

Bacterial expression of Fc-L-ShK[1-35] inhibitor of Kv1.3. The methodsto clone and express the peptibody in bacteria are described above. Thevector used was pAMG21ampR-Fc-Pep and the oligos listed below were usedto generate a duplex (see below) for cloning and expression in bacteriaof Fc-L-ShK[1-35].

Oligos used to form the duplex:

TGGTTCCGGTGGTGGTGGTTCCCGTTCCTGCATCGACACCAT; //SEQ ID NO: 669CCCGAAATCCCGTTGCACCGCTTTCCAGTGCAAACACTCCATGAAATACCGTCTGTCCTTCTGCCGTAAAACC//SEQ ID NO: 670 TGCGGTACCTGC;CTTAGCAGGTACCGCAGGTTTTACGGCAGAAGGACAGACGGT; //SEQ ID NO: 671ATTTCATGGAGTGTTTGCACTGGAAAGCGGTGCAACGGGATTTCGGGATGGTGTCGATGCAGGAACGGGAACC//SEQ ID NO: 672 ACCACCACCGGA;

The oligo duplex is shown below:

TGGTTCCGGTGGTGGTGGTTCCCGTTCCTGCATCGACACCATCCCGAAATCCCGTTGCAC//SEQ ID NO: 673 1---------+---------+---------+---------+---------+---------+  60    AGGCCACCACCACCAAGGGCAAGGACGTAGCTGTGGTAGGGCTTTAGGGCAACGTG//SEQ ID NO: 675 G  S  G  G  G  G  S  R  S  C  I  D  T  I  P  K  S  R  C  T −//SEQ ID NO: 674CGCTTTCCAGTGCAAACACTCCATGAAATACCGTCTGTCCTTCTGCCGTAAAACCTGCGG 61---------+---------+---------+---------+---------+---------+ 120GCGAAAGGTCACGTTTGTGAGGTACTTTATGGCAGACAGGAAGACGGCATTTTGGACGCC A  F  Q  C  K  H  S  M  K  Y  R  L  S  F  C  R  K  T  C  G − TACCTGC121 ------- ATGGACGATTC  T  C   −

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen. Purification of bacterially expressedFc-L10-ShK(1-35) is further described in Example 38 herein below.

Example 4 Fc-L-ShK[2-35] Bacterial Expression

Bacterial expression of Fc-L-ShK[2-35]. The methods to clone and expressthe peptibody in bacteria are described in Example 3. The vector usedwas pAMG21ampR-Fc-Pep and the oligos listed below were used to generatea duplex (see below) for cloning and expression in bacteria ofFc-L-ShK[2-35].

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCTGCATCGACACCATCCCGAAATCCCGTTGCACCGCTTTCCAGTGCAAACACTCCATGAAAT;//SEQ ID NO: 676 ACCGTCTGTCCTTCTGCCGTAAAACCTGCGGTACCTGC;//SEQ ID NO: 677CTTAGCAGGTACCGCAGGTTTTACGGCAGAAGGACAGACGGTATTTCATGGAGTGTTTGCACTGGAAAGCGGTGCAACGGGA;//SEQ ID NO: 678 TTTCGGGATGGTGTCGATGCAGGAACCACCACCACCGGA;//SEQ ID NO: 679 The oligo duplex formed is shown below:TGGTTCCGGTGGTGGTGGTTCCTGCATCGACACCATCCCGAAATCCCGTTGCACCGCTTT//SEQ ID NO: 680 1---------+---------+---------+---------+---------+---------+  60    AGGCCACCACCACCAAGGACGTAGCTGTGGTAGGGCTTTAGGGCAACGTGGCGAAA//SEQ ID NO: 682 G  S  G  G  G  G  S  C  I  D  T  I  P  K  S  R  C  T  A  F -//SEQ ID NO: 681CCAGTGCAAACACTCCATGAAATACCGTCTGTCCTTCTGCCGTAAAACCTGCGGTACCTG 61---------+---------+---------+---------+---------+---------+ 120GGTCACGTTTGTGAGGTACTTTATGGCAGACAGGAAGACGGCATTTTGGACGCCATGGAC Q  C  K  H  S  M  K  Y  R  L  S  F  C  R  K  T  C  G  T  C - C 121 -GATTC

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen. Purification of bacterially expressedFc-L10-ShK(2-35) is further described in Example 39 herein below.

Example 5 Fc-L-HmK Bacterial Expression

Bacterial expression of Fc-L-HmK. The methods to clone and express thepeptibody in bacteria are described in Example 3. The vector used waspAMG21ampR-Fc-Pep and the oligos listed below were used to generate aduplex (see below) for cloning and expression in bacteria of Fc-L-HmK.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCCGTACCTGCAAAGACCTGAT; //SEQ ID NO: 690CCCGGTTTCCGAATGCACCGACATCCGTTGCCGTACCTCCATGAAATACCGTCTGAACCTGTGCCGTAAAACCTGCGGTTCCSEQ ID NO: 692 TGC; CTTAGCAGGAACCGCAGGTTTTACGGCACAGGTTCAGACGGT;//SEQ ID NO: 693 4142-94       ATTTCATGGAGGTACGGCAACGGATGTCGGTGCATTCGGAAACCGGGATCAGGTCTTTGCAGGTACGGGAACCAC//SEQ ID NO: 694 CACCACCGGA

The oligo duplex formed is shown below:

TGGTTCCGGTGGTGGTGGTTCCCGTACCTGCAAAGACCTGATCCCGGTTTCCGAATGCAC//SEQ ID NO: 695 1---------+---------+---------+---------+---------+---------+  60    AGGCCACCACCACCAAGGGCATGGACGTTTCTGGACTAGGGCCAAAGGCTTACGTG//SEQ ID NO: 697 G  S  G  G  G  G  S  R  T  C  K  D  L  I  P  V  S  E  C  T -//SEQ ID NO: 696CGACATCCGTTGCCGTACCTCCATGAAATACCGTCTGAACCTGTGCCGTAAAACCTGCGG 61---------+---------+---------+---------+---------+---------+ 120GCTGTAGGCAACGGCATGGAGGTACTTTATGGCAGACTTGGACACGGCATTTTGGACGCC D  I  R  C  R  T  S  M  K  Y  R  L  N  L  C  R  K  T  C  G - TTCCTGC121 ------- AAGCACGATTC  S  C   -

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 6 Fc-L-KTX1 Bacterial Expression

Bacterial expression of Fc-L-KTX1. The methods to clone and express thepeptibody in bacteria are described in Example 3. The vector used waspAMG21ampR-Fc-Pep and the oligos listed below were used to generate aduplex (see below) for cloning and expression in bacteria of Fc-L-KTX1.

Oligos used to form duplex are shown below

TGGTTCCGGTGGTGGTGGTTCCGGTGTTGAAATCAACGTTAAATGCT; //SEQ ID NO: 698CCGGTTCCCCGCAGTGCCTGAAACCGTGCAAAGACGCTGGTATGCGTTTCGGTAAATGCATGAACCGTAAATGCCACTGCAC//SEQ ID NO: 699 CCCGAAA;CTTATTTCGGGGTGCAGTGGCATTTACGGTTCATGCATTTACCGAAA; //SEQ ID NO: 700CGCATACCAGCGTCTTTGCACGGTTTCAGGCACTGCGGGGAACCGGAGCATTTAACGTTGATTTCAACACCGGAACCACCAC//SEQ ID NO: 701 CACCGGA;

The oligo duplex formed is shown below:

TGGTTCCGGTGGTGGTGGTTCCGGTGTTGAAATCAACGTTAAATGCTCCGGTTCCCCGCA//SEQ ID NO: 702 1---------+---------+---------+---------+---------+---------+  60    AGGCCACCACCACCAAGGCCACAACTTTAGTTGCAATTTACGAGGCCAAGGGGCGT//SEQ ID NO: 704 G  S  G  G  G  G  S  G  V  E  I  N  V  K  C  S  G  S  P  Q -//SEQ ID NO: 703GTGCCTGAAACCGTGCAAAGACGCTGGTATGCGTTTCGGTAAATGCATGAACCGTAAATG 61---------+---------+---------+---------+---------+---------+ 120CACGGACTTTGGCACGTTTCTGCGACCATACGCAAAGCCATTTACGTACTTGGCATTTAC C  L  K  P  C  K  D  A  G  M  R  F  G  K  C  M  N  R  K  C -CCACTGCACCCCGAAA 121 ---------+------ GGTGACGTGGGGCTTTATTC H  C  T  P  K   -

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Purification and refolding of Fc-L-KTX1 expressed in bacteria. Frozen,E. coli paste (28 g) was combined with 210 ml of room temperature 50 mMtris HCl, 5 mM EDTA, pH 8.0 and was brought to about 0.1 mg/ml hen eggwhite lysozyme. The suspended paste was passed through a chilledmicrofluidizer twice at 12,000 PSI. The cell lysate was then centrifugedat 22,000 g for 20 min at 4° C. The pellet was then resuspended in 200ml 1% deoxycholic acid using a tissue grinder and then centrifuged at22,000 g for 20 min at 4° C. The pellet was then resuspended in 200 mlwater using a tissue grinder and then centrifuged at 22,000 g for 20 minat 4° C. The pellet (4.8 g) was then dissolved in 48 ml 8 M guanidineHCl, 50 mM tris HCl, pH 8.0. The dissolved pellet was then reduced byadding 30 μl 1 M dithiothreitol to 3 ml of the solution and incubatingat 37° C. for 30 minutes. The reduced pellet solution was thencentrifuged at 14,000 g for 5 min at room temperature, and then 2.5 mlof the supernatant was transferred to 250 ml of the refolding buffer (2M urea, 50 mM tris, 160 mM arginine HCl, 5 mM EDTA, 1 mM cystamine HCl,4 mM cysteine, pH 8.5) at 4° C. with vigorous stirring. The stirringrate was then slowed and the incubation was continued for 2 days at 4°C. The refolding solution was then filtered through a 0.22 μm celluloseacetate filter and stored at 4° C. for 3 days.

The stored refold was then diluted with 1 L of water and the pH wasadjusted to 7.5 using 1 M H₃PO₄. The pH adjusted material was thenloaded on to a 10 ml Amersham SP-HP HiTrap column at 10 ml/min inS-Buffer A (20 mM NaH₂PO₄, pH 7.3) at 7° C. The column was then washedwith several column volumes of S-Buffer A, followed by elution with alinear gradient from 0% to 60% S-Buffer B (20 mM NaH₂PO₄, 1 M NaCl, pH7.3) followed by a step to 100% S-Buffer B at 5 ml/min 7° C. Fractionswere then analyzed using a Coomassie brilliant blue stained tris-glycine4-20% SDS-PAGE, and the fractions containing the desired product werepooled based on these data (45 ml). The pool was then loaded on to a 1ml Amersham rProtein A HiTrap column in PBS at 2 ml/min 7° C. Thencolumn was then washed with several column volumes of PBS and elutedwith 100 mM glycine pH 3.0. To the elution peak (2.5 ml), 62.5 μl 2 Mtris base was added, and then the pH adjusted material was filteredthough a Pall Life Sciences Acrodisc with a 0.22 μm, 25 mm Mustang Emembrane at 2 ml/min room temperature.

A spectral scan was then conducted on 20 μl of the combined pool dilutedin 700 μl PBS using a Hewlett Packard 8453 spectrophotometer (FIG. 28C).The concentration of the filtered material was determined to be 2.49mg/ml using a calculated molecular mass of 30,504 g/mol and extinctioncoefficient of 35,410 M⁻¹ cm⁻¹. The purity of the filtered material wasthen assessed using a Coomassie brilliant blue stained tris-glycine4-20% SDS-PAGE (FIG. 28A). The endotoxin level was then determined usinga Charles River Laboratories Endosafe-PTS system (0.05-5 EU/mlsensitivity) using a 50-fold dilution of the sample in Charles RiversEndotoxin Specific Buffer BG120 yielding a result of <1 EU/mg protein.The macromolecular state of the product was then determined using sizeexclusion chromatography on 45 μg of the product injected on to aPhenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mMNaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 28B).The product was then subject to mass spectral analysis by diluting 1 μlof the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05%trifluroacetic acid, 50% acetonitrile). The resultant solution (1 μl)was spotted onto a MALDI sample plate. The sample was allowed to drybefore being analyzed using a Voyager DE-RP time-of-flight massspectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). Thepositive ion/linear mode was used, with an accelerating voltage of 25kV. Each spectrum was produced by accumulating data from ˜200 lasershots. External mass calibration was accomplished using purifiedproteins of known molecular masses (FIG. 28D) and these studiesconfirmed the integrity of the purified peptibody, within experimentalerror. The product was then stored at −80° C.

Purified Fc-L-KTX1 blocked the human Kv1.3 current in a dose-dependentfashion (FIG. 32A and FIG. 32B) by electrophysiology (method was asdescribed in Example 36).

Example 7 Fc-L-HsTx1 Bacterial Expression

Bacterial expression of Fc-L-HsT1. The methods to clone and express thepeptibody in bacteria are described in Example 3. The vector used waspAMG21ampR-Fc-Pep and the oligos listed below were used to generate aduplex (see below) for cloning and expression in bacteria of Fc-L-HsTx1.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCGCTTCCTGCCGTACCCCGAAAGAC; //SEQ ID NO: 705TGCGCTGACCCGTGCCGTAAAGAAACCCGTTGCCCGTACGGTAAATGCATGAACCGTAAATGCAAATGCAACC//SEQ ID NO: 706 GTTGC;CTTAGCAACGGTTGCATTTGCATTTACGGTTCATGCATTTACCGTACG; //SEQ ID NO: 707GGCAACCGGTTTCTTTACGGCACGGGTCAGCGCAGTCTTTCGGGGTACGGCAGGAAGCGGAACCACCACCACC//SEQ ID NO: 708 GGA;The duplex formed by the oligos above is shown below:

TGGTTCCGGTGGTGGTGGTTCCGCTTCCTGCCGTACCCCGAAAGACTGCGCTGACCCGTGSEQ ID NO: 709 1---------+---------+---------+---------+---------+---------+  60    AGGCCACCACCACCAAGGCGAAGGACGGCATGGGGCTTTCTGACGCGACTGGGCACSEQ ID NO: 711 G  S  G  G  G  G  S  A  S  C  R  T  P  K  D  C  A  D  P  C -SEQ ID NO: 710CCGTAAAGAAACCGGTTGCCCGTACGGTAAATGCATGAACCGTAAATGCAAATGCAACCG 61---------+---------+---------+---------+---------+---------+ 120GGCATTTCTTTGGCCAACGGGCATGCCATTTACGTACTTGGCATTTACGTTTACGTTGGC R  K  S  T  G  C  P  Y  G  K  C  M  N  R  K  C  K  C  N  R - TTGC 121---- 124 AACGATTC  C   -

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 8 Fc-L-MgTx Bacterial Expression

Bacterial expression of Fc-L-MgTx. The methods to clone and express thepeptibody in bacteria are described in Example 3. The vector used waspAMG21ampR-Fc-Pep and the oligos listed below were used to generate aduplex (see below) for cloning and expression in bacteria of Fc-L-MgTx.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCACCATCATCAACGTTAAATGCACCTC; //SEQ ID NO: 712CCCGAAACAGTGCCTGCCGCCGTGCAAAGCTCAGTTCGGTCAGTCCGCTGGTGCTAAATGCATGAACGGTAAA//SEQ ID NO: 713 TGCAAATGCTACCCGCAC;CTTAGTGCGGGTAGCATTTGCATTTACCGTTCATGCATTTAGCACCAG; //SEQ ID NO: 714CGGACTGACCGAACTGAGCTTTGCACGGCGGCAGGCACTGTTTCGGGGAGGTGCATTTAACGTTGATGATGGT//SEQ ID NO: 715 GGAACCACCACCACCGGA;The oligos above were used to form the duplex shown below:

TGGTTCCGGTGGTGGTGGTTCCACCATCATCAACGTTAAATGCACCTCCCCGAAACAGTGSEQ ID NO: 716 1---------+---------+---------+---------+---------+---------+  60    AGGCCACCACCACCAAGGTGGTAGTAGTTGCAATTTACGTGGAGGGGCTTTGTCACSEQ ID NO: 718 G  S  G  G  G  G  S  T  I  I  N  V  K  C  T  S  P  K  Q  C -SEQ ID NO: 717CCTGCCGCCGTGCAAAGCTCAGTTCGGTCAGTCCGCTGGTGCTAAATGCATGAACGGTAA 61---------+---------+---------+---------+---------+---------+ 120GGACGGCGGCACGTTTCGAGTCAAGCCAGTCAGGCGACCACGATTTACGTACTTGCCATT L  P  P  C  K  A  Q  F  G  Q  S  A  G  A  K  C  M  N  G  K -ATGCAAATGCTACCCGCAC 121 ---------+--------- TACGTTTACGATGGGCGTGATTC C  K  C  Y  P  H   -

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 9 Fc-L-AgTx2 Bacterial Expression

Bacterial expression of Fc-L-AgTx2. The methods to clone and express thepeptibody in bacteria are described in Example 3. The vector used waspAMG21ampR-Fc-Pep and the oligos listed below were used to generate aduplex (see below) for cloning and expression in bacteria of Fc-L-AgTx2.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCGGTGTTCCGATCAACGTTTCCTGCACCGGT; //SEQ ID NO: 719TCCCCGCAGTGCATCAAACCGTGCAAAGACGCTGGTATGCGTTTCGGTAAATGCATGAACCGTAAATGCCACT//SEQ ID NO: 720 GCACCCCGAAA;CTTATTTCGGGGTGCAGTGGCATTTACGGTTCATGCATTTACCGAAACGCATA; //SEQ ID NO: 721CCAGCGTCTTTGCACGGTTTGATGCACTGCGGGGAACCGGTGCAGGAAACGTTGATCGGAACACCGGAACCAC//SEQ ID NO: 722 CACCACCGGA;The oligos listed above were used to form the duplex shown below:

TGGTTCCGGTGGTGGTGGTTCCGGTGTTCCGATCAACGTTTCCTGCACCGGTTCCCCGCASEQ ID NO: 723 1---------+---------+---------+---------+---------+---------+  60    AGGCCACCACCACCAAGGCCACAAGGCTAGTTGCAAAGGACGTGGCCAAGGGGCGTSEQ ID NO: 725 G  S  G  G  G  G  S  G  V  P  I  N  V  S  C  T  G  S  P  Q -SEQ ID NO: 724GTGCATCAAACCGTGCAAAGACGCTGGTATGCGTTTCGGTAAATGCATGAACCGTAAATG 61---------+---------+---------+---------+---------+---------+ 120CACGTAGTTTGGCACGTTTCTGCGACCATACGCAAAGCCATTTACGTACTTGGCATTTAC C  I  K  P  C  K  D  A  G  M  R  F  G  K  C  M  N  R  K  C -CCACTGCACCCCGAAA_(—) 121 ---------+------ GGTGACGTGGGGCTTTATTC H  C  T  P  K   -

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Refolding and purification of Fc-L-AgTx2 expressed in bacteria. Frozen,E. coli paste (15 g) was combined with 120 ml of room temperature 50 mMtris HCl, 5 mM EDTA, pH 8.0 and was brought to about 0.1 mg/ml hen eggwhite lysozyme. The suspended paste was passed through a chilledmicrofluidizer twice at 12,000 PSI. The cell lysate was then centrifugedat 22,000 g for 20 min at 4° C. The pellet was then resuspended in 200ml 1% deoxycholic acid using a tissue grinder and then centrifuged at22,000 g for 20 min at 4° C. The pellet was then resuspended in 200 mlwater using a tissue grinder and then centrifuged at 22,000 g for 20 minat 4° C. The pellet (4.6 g) was then dissolved in 46 ml 8 M guanidineHCl, 50 mM tris HCl, pH 8.0. The dissolved pellet was then reduced byadding 30 μl 1 M dithiothreitol to 3 ml of the solution and incubatingat 37° C. for 30 minutes. The reduced pellet solution was thencentrifuged at 14,000 g for 5 min at room temperature, and then 2.5 mlof the supernatant was transferred to 250 ml of the refolding buffer (2M urea, 50 mM tris, 160 mM arginine HCl, 5 mM EDTA, 1 mM cystamine HCl,4 mM cysteine, pH 9.5) at 4° C. with vigorous stirring. The stirringrate was then slowed and the incubation was continued for 2 days at 4°C. The refolding solution was then filtered through a 0.22 μm celluloseacetate filter and stored at −70° C.

The stored refold was defrosted and then diluted with 1 L of water andthe pH was adjusted to 7.5 using 1 M H₃PO₄. The pH adjusted material wasthen filtered through a 0.22 μm cellulose acetate filter and loaded onto a 10 ml Amersham SP-HP HiTrap column at 10 ml/min in S-Buffer A (20mM NaH₂PO₄, pH 7.3) at 7° C. The column was then washed with severalcolumn volumes of S-Buffer A, followed by elution with a linear gradientfrom 0% to 60% S-Buffer B (20 mM NaH₂PO₄, 1 M NaCl, pH 7.3) followed bya step to 100% S-Buffer B at 5 ml/min 7° C. Fractions were then analyzedusing a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE,and the fractions containing the desired product were pooled based onthese data (15 ml). The pool was then loaded on to a 1 ml AmershamrProtein A HiTrap column in PBS at 2 ml/min 7° C. Then column was thenwashed with several column volumes of 20 mM NaH₂PO₄ pH 6.5, 1 M NaCl andeluted with 100 mM glycine pH 3.0. To the elution peak (1.5 ml), 70 μl 1M tris HCl pH 8.5 was added, and then the pH-adjusted material wasfiltered though a 0.22 μm cellulose acetate filter.

A spectral scan was then conducted on 20 μl of the combined pool dilutedin 700 μl PBS using a Hewlett Packard 8453 spectrophotometer (FIG. 29C).The concentration of the filtered material was determined to be 1.65mg/ml using a calculated molecular mass of 30,446 g/mol and extinctioncoefficient of 35,410 M⁻¹ cm⁻¹. The purity of the filtered material wasthen assessed using a Coomassie brilliant blue stained tris-glycine4-20% SDS-PAGE (FIG. 29A). The endotoxin level was then determined usinga Charles River Laboratories Endosafe-PTS system (0.05-5 EU/mlsensitivity) using a 33-fold dilution of the sample in Charles RiversEndotoxin Specific Buffer BG120 yielding a result of <4 EU/mg protein.The macromolecular state of the product was then determined using sizeexclusion chromatography on 20 μg of the product injected on to aPhenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mMNaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 29D).The product was then subject to mass spectral analysis by diluting 1 μlof the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05%trifluroacetic acid, 50% acetonitrile). The resultant solution (1 μl)was spotted onto a MALDI sample plate. The sample was allowed to drybefore being analyzed using a Voyager DE-RP time-of-flight massspectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). Thepositive ion/linear mode was used, with an accelerating voltage of 25kV. Each spectrum was produced by accumulating data from ˜200 lasershots. External mass calibration was accomplished using purifiedproteins of known molecular masses (FIG. 29E) and these studiesconfirmed the integrity of the purified peptibody, within experimentalerror. The product was then stored at −80° C.

Example 10 Fc-L-OSK1 Bacterial Expression

Bacterial expression of Fc-L-OSK1. The methods used to clone and expressthe peptibody in bacteria were as described in Example 3. The vectorused was pAMG21ampR-Fc-Pep and the oligos listed below were used togenerate a duplex (see below) for cloning and expression in bacteria ofFc-L-OSK1.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCGGTGTTATCATCAACGTTAAATGCAAAATCTCCCGTCAGTGCCTGGAACCG//SEQ ID NO: 726 TGCAAAAAAG;CTGGTATGCGTTTCGGTAAATGCATGAACGGTAAATGCCACTGCACCCCGAAA; //SEQ ID NO: 727CTTATTTCGGGGTGCAGTGGCATTTACCGTTCATGCATTTACCGAAACGCATACCAGCTTTTTTGCACGGTTC//SEQ ID NO: 728 CAGGCACTGA;CGGGAGATTTTGCATTTAACGTTGATGATAACACCGGAACCACCACCACCGGA; //SEQ ID NO: 729The oligos shown above were used to form the duplex below:

TGGTTCCGGTGGTGGTGGTTCCGGTGTTATCATCAACGTTAAATGCAAAATCTCCCGTCASEQ ID NO: 730 1---------+---------+---------+---------+---------+---------+  60    AGGCCACCACCACCAAGGCCACAATAGTAGTTGCAATTTACGTTTTAGAGGGCAGTSEQ ID NO: 732 G  S  G  G  G  G  S  G  V  I  I  N  V  K  C  K  I  S  R  Q -SEQ ID NO: 731GTGCCTGGAACCGTGCAAAAAAGCTGGTATGCGTTTCGGTAAATGCATGAACGGTAAATG 61---------+---------+---------+---------+---------+---------+ 120CACGGACCTTGGCACGTTTTTTCGACCATACGCAAAGCCATTTACGTACTTGCCATTTAC C  L  E  P  C  K  K  A  G  M  R  F  G  K  C  N  N  G  K  C -CCACTGCACCCCGAAA 121 ---------+------ GGTGACGTGGGGCTTTATTC H  C  T  P  K   -

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen for later use. Purification of Fc-L10-OSK1 fromE. coli paste is described in Example 40 herein below.

Example 11 Fc-L-OSK1(E16K,K20D) Bacterial Expression

Bacterial expression of Fc-L-OSK1(E16K,K20D). The methods to clone andexpress the peptibody in bacteria are described in Example 3. The vectorused was pAMG21ampR-Fc-Pep and the oligos listed below were used togenerate a duplex (see below) for cloning and expression in bacteria ofFc-L-OSK1(E16K,K20D).

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCGGTGTTATCATCAACGTTAAATGCAAAATCTCCCGTCAGTGCCTGAAACCG//SEQ ID NO: 733 TGCAAAGACG;CTGGTATGCGTTTCGGTAAATGCATGAACGGTAAATGCCACTGCACCCCGAAA; //SEQ ID NO: 734CTTATTTCGGGGTGCAGTGGCATTTACCGTTCATGCATTTACCGAAACGCATACCAGCGTCTTTGCACGGTTT//SEQ ID NO: 735 CAGGCACTGA;CGGGAGATTTTGCATTTAACGTTGATGATAACACCGGAACCACCACCACCGGA; //SEQ ID NO: 736

The oligos shown above were used to form the duplex below:

TGGTTCCGGTGGTGGTGGTTCCGGTGTTATCATCAACGTTAAATGCAAAATCTCCCGTCASEQ ID NO: 737 1---------+---------+---------+---------+---------+---------+  60    AGGCCACCACCACCAAGGCCACAATAGTAGTTGCAATTTACGTTTTAGAGGGCAGTSEQ ID NO: 739 G  S  G  G  G  G  S  G  V  I  I  N  V  K  C  K  I  S  R  Q -SEQ ID NO: 738GTGCCTGAAACCGTGCAAAGACGCTGGTATGCGTTTCGGTAAATGCATGAACGGTAAATG 61---------+---------+---------+---------+---------+---------+ 120CACGGACTTTGGCACGTTTCTGCGACCATACGCAAAGCCATTTACGTACTTGCCATTTAC C  L  K  P  C  K  D  A  G  M  R  F  G  K  C  M  N  G  K  C -CCACTGCACCCCGAAA 121 ---------+------ GGTGACGTGGGGCTTTATTC H  C  T  P  K   -

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen for later use.

Example 12 Fc-L-Anuroctoxin Bacterial Expression

Bacterial expression of Fc-L-Anuroctoxin. The methods to clone andexpress the peptibody in bacteria are described in Example 3. The vectorused was pAMG21ampR-Fc-Pep and the oligos listed below were used togenerate a duplex (see below) for cloning and expression in bacteria ofFc-L-Anuroctoxin.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCAAAGAATGCACCGGTCCGCAGCACTGCACCAACTTCTGCCGTAAAAACAAA//SEQ ID NO: 740 TGCACCCACG; GTAAATGCATGAACCGTAAATGCAAATGCTTCAACTGCAAA;//SEQ ID NO: 741CTTATTTGCAGTTGAAGCATTTGCATTTACGGTTCATGCATTTACCGTGGGTGCATTTGTTTTTACGGCAGAA//SEQ ID NO: 742 GTTGGTGCAG; TGCTGCGGACCGGTGCATTCTTTGGAACCACCACCACCGGA;//SEQ ID NO: 743The oligos shown above were used to form the duplex below:

TGGTTCCGGTGGTGGTGGTTCCAAAGAATGCACCGGTCCGCAGCACTGCACCAACTTCTGSEQ ID NO: 744 1---------+---------+---------+---------+---------+---------+ 60    AGGCCACCACCACCAAGGTTTCTTACGTGGCCAGGCGTCGTGACGTGGTTGAAGACSEQ ID NO: 746 G  S  G  G  G  G  S  K  E  C  T  G  P  Q  H  C  T  N  F  C  -SEQ ID NO: 745CCGTAAAAACAAATGCACCCACGGTAAATGCATGAACCGTAAATGCAAATGCTTCAACTG 61---------+---------+---------+---------+---------+---------+ 120GGCATTTTTGTTTACGTGGGTGCCATTTACGTACTTGGCATTTACGTTTACGAAGTTGAC R  K  N  K  C  T  H  G  K  C  M  N  R  K  C  K  C  F  N  C  - CAAA 121---- GTTTATTC  K   -

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 13 Fc-L-Noxiustoxin Bacterial Expression

Bacterial expression of Fc-L-Noxiustoxin or Fc-L-NTX. The methods toclone and express the peptibody in bacteria are described in Example 3.The vector used was pAMG21ampR-Fc-Pep and the oligos listed below wereused to generate a duplex (see below) for cloning and expression inbacteria of Fc-L-NTX.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCACCATCATCAACGTTAAATGCACCTCCCCGAAACAGTGCTCCAAACCGTGC//SEQ ID NO: 747 AAAGAACTGT;ACGGTTCCTCCGCTGGTGCTAATGCATGAACGGTAAATGCAAATGCTACAACAAC;//SEQ ID NO: 748CTTAGTTGTTGTAGCATTTGCATTTACCGTTCATGCATTTAGCACCAGCGGAGGAACCGTACAGTTCTTTGCA//SEQ ID NO: 749 CGGTTTGGAG;CACTGTTTCGGGGAGGTGCATTTAACGTTGATGATGGTGGAACCACCACCACCGGA;//SEQ ID NO: 750The oligos shown above were used to form the duplex below:

TGGTTCCGGTGGTGGTGGTTCCACCATCATCAACGTTAAATGCACCTCCCCGAAACAGTGSEQ ID NO: 751 1---------+---------+---------+---------+---------+---------+ 60    AGGCCACCACCACCAAGGTGGTAGTAGTTGCAATTTACGTGGAGGGGCTTTGTCACSEQ ID NO: 753 G  S  G  G  G  G  S  T  I  I  N  V  K  C  T  S  P  K  Q  C  -SEQ ID NO: 752CTCCAAACCGTGCAAAGAACTGTACGGTTCCTCCGCTGGTGCTAAATGCATGAACGGTAA 61---------+---------+---------+---------+---------+---------+ 120GAGGTTTGGCACGTTTCTTGACATGCCAAGGAGGCGACCACGATTTACGTACTTGCCATT S  K  P  C  K  E  L  Y  G  S  S  A  G  A  K  C  M  N  G  K  -ATGCAAATGCTACAACAAC 121 ---------+--------- TACGTTTACGATGTTGTTGATTC C  K  C  Y  N  N   -

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 14 Fc-L-Pi2 Bacterial Expression

Bacterial expression of Fc-L-Pi2. The methods to clone and express thepeptibody in bacteria are described in Example 3. The vector used waspAMG21ampR-Fc-Pep and the oligos listed below were used to generate aduplex (see below) for cloning and expression in bacteria of Fc-L-Pi2.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCACCATCTCCTGCACCAACCCG; //SEQ ID NO: 754AAACAGTGCTACCCGCACTGCAAAAAAGAAACCGGTTACCCGAACGCTAAATGCATGAACCGTAAATGCAAAT//SEQ ID NO: 755 GCTTCGGTCGT;CTTAACGACCGAAGCATTTGCATTTACGGTTCATGCATTTAGCG; //SEQ ID NO: 756TTCGGGTAACCGGTTTCTTTTTTGCAGTGCGGGTAGCACTGTTTCGGGTTGGTGCAGGAGATGGTGGAACCAC//SEQ ID NO: 757 CACCACCGGA;The oligos above were used to form the duplex below:

TGGTTCCGGTGGTGGTGGTTCCACCATCTCCTGCACCAACCCGAAACAGTGCTACCCGCASEQ ID NO: 758 1---------+---------+---------+---------+---------+---------+ 60    AGGCCACCACCACCAAGGTGGTAGAGGACGTGGTTGGGCTTTGTCACGATGGGCGTSEQ ID NO: 760 G  S  G  G  G  G  S  T  I  S  C  T  N  P  K  Q  C  Y  P  H  -SEQ ID NO: 759CTGCAAAAAAGAAACCGGTTACCCGAACGCTAAATGCATGAACCGTAAATGCAAATGCTT 61---------+---------+---------+---------+---------+---------+ 120GACGTTTTTTCTTTGGCCAATGGGCTTGCGATTTACGTACTTGGCATTTACGTTTACGAA C  K  K  E  T  G  Y  P  N  A  K  C  M  N  R  K  C  K  C  F  - CGGTCGT121 ------- GCCAGCAATTC  G  R   -

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 15 ShK[1-35]-L-Fc Bacterial Expression

Bacterial expression of ShK[1-35]-L-Fc. The methods to clone and expressthe peptibody in bacteria are described in Example 3. The vector usedwas pAMG21ampR-Pep-Fc and the oligos listed below were used to generatea duplex (see below) for cloning and expression in bacteria ofShK[1-35]-L-Fc.

Oligos used to form duplex are shown below:

TATGCGTTCTTGTATTGATACTATTCCAAAATCTCGTTGTACTGCTTTTCAATGTAAACATTCTATGAAATAT//SEQ ID NO: 761 CGTCTTTCTT;TTTGTCGTAAAACTTGTGGTACTTGTTCTGGTGGTGGTGGTTCT; //SEQ ID NO: 762CACCAGAACCACCACCACCAGAACAAGTACCACAAGTTTTACGACAAAAAGAAAGACGATATTTCATAGAATG//SEQ ID NO: 763 TTTACATTGA;AAAGCAGTACAACGAGATTTTGGAATAGTATCAATACAAGAACG; //SEQ ID NO: 764The oligos shown above were used to form the duplex shown below:

TATGCGTTCTTGTATTGATACTATTCCAAAATCTCGTTGTACTGCTTTTCAATGTAAACASEQ ID NO: 765 1---------+---------+---------+---------+---------+---------+ 60    GCAAGAACATAACTATGATAAGGTTTTAGAGCAACATGACGAAAAGTTACATTTGTSEQ ID NO: 767 M  R  S  C  I  D  T  I  P  K  S  R  C  T  A  F  Q  C  K  H  -SEQ ID NO: 766TTCTATGAAATATCGTCTTTCTTTTTGTCGTAAAACTTGTGGTACTTGTTCTGGTGGTGG 61---------+---------+---------+---------+---------+---------+ 120AAGATACTTTATAGCAGAAAGAAAAACAGCATTTTGAACACCATGAACAAGACCACCACC S  M  K  Y  R  L  S  F  C  R  K  T  C  G  T  C  S  G  G  G  - TGGTTCT121 ------- 127 ACCAAGACCAC  G  S   -

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen. Purification of met-ShK[1-35]-Fc was asdescribed in Example 51 herein below.

Example 16 ShK[2-35]-L-Fc Bacterial Expression

Bacterial expression of ShK[2-35]-L-Fc. The methods to clone and expressthe peptibody in bacteria are described in Example 3. The vector usedwas pAMG21ampR-Pep-Fc and the oligos listed below were used to generatea duplex (see below) for cloning and expression in bacteria ofShK[2-35]-L-Fc.

Oligos used to form duplex are shown below:

TATGTCTTGTATTGATACTATTCCAAAATCTCGTTGTACTGCTTTTCAATGTAAACATTCTATGAAATATCGT//SEQ ID NO: 768 CTTTCTT; TTTGTCGTAAAACTTGTGGTACTTGTTCTGGTGGTGGTGGTTCT;//SEQ ID NO: 769CACCAGAACCACCACCACCAGAACAAGTACCACAAGTTTTACGACAAAAAGAAAGACGATATTTCATAGAATG//SEQ ID NO: 770 TTTACATTGA; AAAGCAGTACAACGAGATTTTGGAATAGTATCAATACAAGA;SEQ ID NO: 771The oligos above were used to form the duplex shown below:

TATGTCTTGTATTGATACTATTCCAAAATCTCGTTGTACTGCTTTTCAATGTAAACATTCSEQ ID NO: 772 1---------+---------+---------+---------+---------+---------+ 60    AGAACATAACTATGATAAGGTTTTAGAGCAACATGACGAAAAGTTACATTTGTAAGSEQ ID NO: 774 M  S  C  I  D  T  I  P  K  S  R  C  T  A  F  Q  C  K  H  S  -SEQ ID NO: 773TATGAAATATCGTCTTTCTTTTTGTCGTAAAACTTGTGGTACTTGTTCTGGTGGTGGTGG 61---------+---------+---------+---------+---------+---------+ 120ATACTTTATAGCAGAAAGAAAAACAGCATTTTGAACACCATGAACAAGACCACCACCACC M  K  Y  R  L  S  F  C  R  K  T  C  G  T  C  S  G  G  G  G  - TTCT 121---- AAGACCAC  S   -

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen. Purification of the ShK[2-35]-Fc was asdescribed in Example 50 herein below.

Example 17 Fc-L-ChTx Bacterial Expression

Bacterial expression of Fc-L-ChTx. The methods to clone and express thepeptibody in bacteria are described in Example 3. The vector used waspAMG21ampR-Fc-Pep and the oligos listed below were used to generate aduplex (see below) for cloning and expression in bacteria of Fc-L-ChTx.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCCAGTTCACCAACGTT; //SEQ ID NO: 775TCCTGCACCACCTCCAAAGAATGCTGGTCCGTTTGCCAGCGTCTGCACAACACCTCCCGTGGTAAATGCATGA//SEQ ID NO: 776 ACAAAAAATGCCGTTGCTACTCC;CTTAGGAGTAGCAACGGCATTTTTTGTTCATGCATTTA; //SEQ ID NO: 777CCACGGGAGGTGTTGTGCAGACGCTGGCAAACGGACCAGCATTCTTTGGAGGTGGTGCAGGAAACGTTGGTGA//SEQ ID NO: 778 ACTGGGAACCACCACCACCGGA;The oligos shown above were used to form the duplex below:

TGGTTCCGGTGGTGGTGGTTCCCAGTTCACCAACGTTTCCTGCACCACCTCCAAAGAATGSEQ ID NO: 779 1---------+---------+---------+---------+---------+---------+ 60    AGGCCACCACCACCAAGGGTCAAGTGGTTGCAAAGGACGTGGTGGAGGTTTCTTACSEQ ID NO: 781 G  S  G  G  G  G  S  Q  F  T  N  V  S  C  T  T  S  K  E  C  -SEQ ID NO: 780CTGGTCCGTTTGCCAGCGTCTGCACAACACCTCCCGTGGTAAATGCATGAACAAAAAATG 61---------+---------+---------+---------+---------+---------+ 120GACCAGGCAAACGGTCGCAGACGTGTTGTGGAGGGCACCATTTACGTACTTGTTTTTTAC W  S  V  C  Q  R  L  H  N  T  S  R  G  K  C  M  N  K  K  C  -CCGTTGCTACTCC 121 ---------+--- GGCAACGATGAGGATTC  R  C  Y  S   -

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 18 Fc-L-MTX Bacterial Expression

Bacterial expression of Fc-L-MTX. The methods to clone and express thepeptibody in bacteria are described in Example 3. The vector used waspAMG21ampR-Fc-Pep and the oligos listed below were used to generate aduplex (see below) for cloning and expression in bacteria of Fc-L-MTX.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCGTTTCCTGCACCGGT; //SEQ ID NO: 782TCCAAAGACTGCTACGCTCCGTGCCGTAAACAGACCGGTTGCCCGAACGCTAAATGCATCAACAAATCCTGCA//SEQ ID NO: 783 AATGCTACGGTTGC; CTTAGCAACCGTAGCATTTGCAGGATTTGTTGATGCAT;//SEQ ID NO: 784TTAGCGTTCGGGCAACCGGTCTGTTTACGGCACGGAGCGTAGCAGTCTTTGGAACCGGTGCAGGAAACGGAAC//SEQ ID NO: 785 CACCACCACCGGA;The oligos above were used to form the duplex shown below:

TGGTTCCGGTGGTGGTGGTTCCGTTTCCTGCACCGGTTCCAAAGACTGCTACGCTCCGTGSEQ ID NO: 786 1---------+---------+---------+---------+---------+---------+ 60    AGGCCACCACCACCAAGGCAAAGGACGTGGCCAAGGTTTCTGACGATGCGAGGCACSEQ ID NO: 788 G  S  G  G  G  G  S  V  S  C  T  G  S  K  D  C  Y  A  P  C  -SEQ ID NO: 787CCGTAAACAGACCGGTTGCCCGAACGCTAAATGCATCAACAAATCCTGCAAATGCTACGG 61---------+---------+---------+---------+---------+---------+ 120GGCATTTGTCTGGCCAACGGGCTTGCGATTTACGTAGTTGTTTAGGACGTTTACGATGCC R  K  Q  T  G  C  P  N  A  K  C  I  N  K  S  C  K  C  Y  G  - TTGC 121---- AACGATTC  C   -

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 19 Fc-L-ChTx(K32E) Bacterial Expression

Bacterial expression of Fc-L-ChTx(K32E). The methods to clone andexpress the peptibody in bacteria are described in Example 3. The vectorused was pAMG21ampR-Fc-Pep and the oligos listed below were used togenerate a duplex (see below) for cloning and expression in bacteria ofFc-L-ChTx(K32E).

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCCAGTTCACCAACGTTTCCTG; //SEQ ID NO: 789CACCACCTCCAAAGAATGCTGGTCCGTTTGCCAGCGTCTGCACAACACCTCCCGTGGTAAATGCATGAACAAA//SEQ ID NO: 790 GAATGCCGTTGCTACTCC;CTTAGGAGTAGCAACGGCATTCTTTGTTCATGCATTTACCACG; //SEQ ID NO: 791GGAGGTGTTGTGCAGACGCTGGCAAACGGACCAGCATTCTTTGGAGGTGGTGCAGGAAACGTTGGTGAACTGG//SEQ ID NO: 792 GAACCACCACCACCGGA;The oligos shown above were used to form the duplex below:

TGGTTCCGGTGGTGGTGGTTCCCAGTTCACCAACGTTTCCTGCACCACCTCCAAAGAATGSEQ ID NO: 793 1---------+---------+---------+---------+---------+---------+ 60    AGGCCACCACCACCAAGGGTCAAGTGGTTGCAAAGGACGTGGTGGAGGTTTCTTACSEQ ID NO: 795 G  S  G  G  G  G  S  Q  F  T  N  V  S  C  T  T  S  K  E  C  -SEQ ID NO: 794CTGGTCCGTTTGCCAGCGTCTGCACAACACCTCCCGTGGTAAATCCATGAACAAAGAATG 61---------+---------+---------+---------+---------+---------+ 120GACCAGGCAAACGGTCGCAGACGTGTTGTGGAGGGCACCATTTACGTACTTGTTTCTTAC W  S  V  C  Q  R  L  H  N  T  S  R  G  K  C  M  N  K  E  C  -CCGTTGCTACTCC 121 ---------+--- GGCAACGATGAGGATTC  R  C  Y  S   -

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 20 Fc-L-Apamin Bacterial Expression

Bacterial expression of Fc-L-Apamin. The methods to clone and expressthe peptibody in bacteria are described in Example 3. The vector usedwas pAMG21ampR-Fc-Pep and the oligos listed below were used to generatea duplex (see below) for cloning and expression in bacteria ofFc-L-Apamin.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCTGCAACTGCAAAGCTCCGGAAACCGCTCTGTGCGCTCGTCGTTGC//SEQ ID NO: 796 CAGCAGCACGGT;CTTAACCGTGCTGCTGGCAACGACGAGCGCACAGAGCGGTTTCCGGAGCTTTGCAGTTGCAGGAACC//SEQ ID NO: 797 ACCACCACCGGA;The oligos above were used to form the duplex shown below:TGGTTCCGGTGGTGGTGGTTCCTGCAACTGCAAAGCTCCGGAAACCGCTCTGTGCGCTCGSEQ ID NO: 798 1---------+---------+---------+---------+---------+---------+ 60    AGGCCACCACCACCAACGACGTTGACGTTTCGAGGCCTTTGGCGACACACGCGAGCSEQ ID NO: 800 G  S  G  G  G  G  S  C  N  C  K  A  P  E  T  A  L  C  A  R  -SEQ ID NO: 799 TCGTTGCCAGCAGCACGGT 61 ---------+---------AGCAACGGTCGTCGTGCCAATTC  R  C  Q  Q  H  G   -

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 21 Fc-L-Scyllatoxin Bacterial Expression

Bacterial expression of Fc-L-Scyllatoxin or Fc-L-ScyTx. The methods toclone and express the peptibody in bacteria are described in Example 3.The vector used was pAMG21ampR-Fc-Pep and the oligos listed below wereused to generate a duplex (see below) for cloning and expression inbacteria of Fc-L-ScyTx.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCGCTTTCTGCAACCTGCG; //SEQ ID NO: 801TATGTGCCAGCTGTCCTGCCGTTCCCTGGGTCTGCTGGGTAAATGCATCGGTGACAAATGCGAATGCGTTAAA//SEQ ID NO: 802 CAC; CTTAGTGTTTAACGCATTCGCATTTGTCACCGATGCATTT;//SEQ ID NO: 803ACCCAGCAGACCCAGGGAACGGCAGGACAGCTGGCACATACGCAGGTTGCAGAAAGCGGAACCACCACCACCG//SEQ ID NO: 804 GA;The oligos above were used to form the duplex below:

TGGTTCCGGTGGTGGTGGTTCCGCTTTCTGCAACCTGCGTATGTGCCAGCTGTCCTGCCGSEQ ID NO: 805 1---------+---------+---------+---------+---------+---------+ 60    AGGCCACCACCACCAAGGCGAAAGACGTTGGACGCATACACGGTCGACAGGACGGCSEQ ID NO: 807 G  S  G  G  G  G  S  A  F  C  N  L  R  M  C  Q  L  S  C  R  -SEQ ID NO: 806 TTCCCTGGGTCTGCTGGGTAAATGCATCGGTGACAAATGCGAATGCGTTAAACAC61 ---------+---------+---------+---------+---------+-----AAGGGACCCAGACGACCCATTTACGTAGCCACTGTTTACGCTTACGCAATTTGTGATTC S  L  G  L  L  G  K  C  I  G  D  K  C  E  C  V  K  H   -

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 22 Fc-L-IbTx Bacterial Expression

Bacterial expression of Fc-L-IbTx. The methods to clone and express thepeptibody in bacteria are described in Example 3. The vector used waspAMG21ampR-Fc-Pep and the oligos listed below were used to generate aduplex (see below) for cloning and expression in bacteria of Fc-L-IbTx.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCCAGTTCACCGACGTTGACTGCTCCGT; //SEQ ID NO: 808TTCCAAAGAATGCTGGTCCGTTTGCAAAGACCTGTTCGGTGTTGACCGTGGTAAATGCATGGGTAAAAAATGC//SEQ ID NO: 809 CGTTGCTACCAG;CTTACTGGTAGCAACGGCATTTTTTACCCATGCATTTACCACGGTCAA; //SEQ ID NO: 810CACCGAACAGGTCTTTGCAAACGGACCAGCATTCTTTGGAAACGGAGCAGTCAACGTCGGTGAACTGGGAACC//SEQ ID NO: 811 ACCACCACCGGA;The oligos above were used to form the duplex below:

TGGTTCCGGTGGTGGTGGTTCCCAGTTCACCGACGTTGACTGCTCCGTTTCCAAAGAATGSEQ ID NO: 812 1---------+---------+---------+---------+---------+---------+ 60    AGGCCACCACCACCAAGGGTCAAGTGGCTGCAACTGACGAGGCAAAGGTTTCTTACSEQ ID NO: 814 G  S  G  G  G  G  S  Q  F  T  D  V  D  C  S  V  S  K  E  C  -SEQ ID NO: 813CTGGTCCGTTTGCAAAGACCTGTTCGGTGTTGACCGTGGTAAATGCATGGGTAAAAAATG 61---------+---------+---------+---------+---------+---------+ 120GACGAGGCAAACGTTTCTGGACAAGCCACAACTGGCACCATTTACGTACCCATTTTTTAC W  S  V  C  K  D  L  F  G  V  D  R  G  K  C  M  G  K  K  C  -CCGTTGCTACCAG 121 ---------+--- GGCAACGATGGTCATTC  R  C  Y  Q   -

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 23 Fc-L-HaTx1 Bacterial Expression

Bacterial expression of Fc-L-HaTx1. The methods to clone and express thepeptibody in bacteria are described in Example 3. The vector used waspAMG21ampR-Fc-Pep and the oligos listed below were used to generate aduplex (see below) for cloning and expression in bacteria of Fc-L-HaTx1.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCGAATGCCGTTACCTGTTCGGTGGTTG; //SEQ ID NO: 815CAAAACCACCTCCGACTGCTGCAAACACCTGGGTTGCAAATTCCGTGACAAATACTGCGCTTGGGACTTCACC//SEQ ID NO: 816 TTCTCC;CTTAGGAGAAGGTGAAGTCCCAAGCGCAGTATTTGTCACGGAATTTGC; //SEQ ID NO: 817AACCCAGGTGTTTGCAGCAGTCGGAGGTGGTTTTGCAACCACCGAACAGGTAACGGCATTCGGAACCACCACC//SEQ ID NO: 818 ACCGGA;The oligos above were used to form the duplex below:

TGGTTCCGGTGGTGGTGGTTCCGAATGCCGTTACCTGTTCGGTGGTTGCAAAACCACCTCSEQ ID NO: 819 1---------+---------+---------+---------+---------+---------+ 60    AGGCCACCACCACCAAGGCTTACGGCAATGGACAAGCCACCAACGTTTTGGTGGAGSEQ ID NO: 821 G  S  G  G  G  G  S  E  C  R  Y  L  F  G  G  C  K  T  T  S  -SEQ ID NO: 820CGACTGCTGCAAACACCTGGGTTGCAAATTCCGTGACAAATACTGCGCTTGGGACTTCAC 61---------+---------+---------+---------+---------+---------+ 120GCTGACGACGTTTGTGGACCCAACGTTTAAGGCACTGTTTATGACGCGAACCCTGAAGTG D  C  C  K  H  L  G  C  K  F  R  D  K  Y  C  A  W  D  F  T  - CTTCTCC121 ------- GAAGAGGATTC  F  S   -

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Refolding and purification of Fc-L-HaTx1 expressed in bacteria. Frozen,E. coli paste (13 g) was combined with 100 ml of room temperature 50 mMtris HCl, 5 mM EDTA, pH 8.0 and was brought to about 0.1 mg/ml hen eggwhite lysozyme. The suspended paste was passed through a chilledmicrofluidizer twice at 12,000 PSI. The cell lysate was then centrifugedat 22,000 g for 20 min at 4° C. The pellet was then resuspended in 200ml 1% deoxycholic acid using a tissue grinder and then centrifuged at22,000 g for 20 min at 4° C. The pellet was then resuspended in 200 mlwater using a tissue grinder and then centrifuged at 22,000 g for 20 minat 4° C. The pellet (2.6 g) was then dissolved in 26 ml 8 M guanidineHCl, 50 mM tris HCl, pH 8.0. The dissolved pellet was then reduced byadding 30 μl 1 M dithiothreitol to 3 ml of the solution and incubatingat 37° C. for 30 minutes. The reduced pellet solution was thencentrifuged at 14,000 g for 5 min at room temperature, and then 2.5 mlof the supernatant was transferred to 250 ml of the refolding buffer (2M urea, 50 mM tris, 160 mM arginine HCl, 5 mM EDTA, 1 mM cystamine HCl,4 mM cysteine, pH 8.5) at 4° C. with vigorous stirring. The stirringrate was then slowed and the incubation was continued for 2 days at 4°C. The refolding solution was then filtered through a 0.22 μm celluloseacetate filter and stored at −70° C.

The stored refold was defrosted and then diluted with 1 L of water andthe pH was adjusted to 7.5 using 1 M H₃PO₄. The pH adjusted material wasthen filtered through a 0.22 μm cellulose acetate filter and loaded onto a 10 ml Amersham SP-HP HiTrap column at 10 ml/min in S-Buffer A (20mM NaH₂PO₄, pH 7.3) at 7° C. The column was then washed with severalcolumn volumes of S-Buffer A, followed by elution with a linear gradientfrom 0% to 60% S-Buffer B (20 mM NaH₂PO₄, 1 M NaCl, pH 7.3) followed bya step to 100% S-Buffer B at 5 ml/min 7° C. Fractions were then analyzedusing a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE,and the fractions containing the desired product were pooled based onthese data (15 ml). The pool was then loaded on to a 1 ml AmershamrProtein A HiTrap column in PBS at 2 ml/min 7° C. Then column was thenwashed with several column volumes of 20 mM NaH₂PO₄ pH 6.5, 1 M NaCl andeluted with 100 mM glycine pH 3.0. To the elution peak (1.4 ml), 70 μl 1M tris HCl pH 8.5 was added, and then the pH adjusted material wasfiltered though a 0.22 μm cellulose acetate filter.

A spectral scan was then conducted on 20 μl of the combined pool dilutedin 700 μl PBS using a Hewlett Packard 8453 spectrophotometer (FIG. 29F).The concentration of the filtered material was determined to be 1.44mg/ml using a calculated molecular mass of 30,469 g/mol and extinctioncoefficient of 43,890 M⁻¹ cm⁻¹. The purity of the filtered material wasthen assessed using a Coomassie brilliant blue stained tris-glycine4-20% SDS-PAGE (FIG. 29B). The endotoxin level was then determined usinga Charles River Laboratories Endosafe-PTS system (0.05-5 EU/mlsensitivity) using a 33-fold dilution of the sample in Charles RiversEndotoxin Specific Buffer BG120 yielding a result of <4 EU/mg protein.The macromolecular state of the product was then determined using sizeexclusion chromatography on 20 μg of the product injected on to aPhenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mMNaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 29G).The product was then subject to mass spectral analysis by diluting 1 μlof the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05%trifluroacetic acid, 50% acetonitrile). The resultant solution (1 μl)was spotted onto a MALDI sample plate. The sample was allowed to drybefore being analyzed using a Voyager DE-RP time-of-flight massspectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). Thepositive ion/linear mode was used, with an accelerating voltage of 25kV. Each spectrum was produced by accumulating data from ˜200 lasershots. External mass calibration was accomplished using purifiedproteins of known molecular masses (FIG. 29H) and these studiesconfirmed the integrity of the purified peptibody, within experimentalerror. The product was then stored at −80° C.

Example 24 Fc-L-PaTx2 Bacterial Expression

Bacterial expression of Fc-L-PaTx2. The methods to clone and express thepeptibody in bacteria are described in Example 3. The vector used waspAMG21ampR-Fc-Pep and the oligos listed below were used to generate aduplex (see below) for cloning and expression in bacteria of Fc-L-PaTx2.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCTACTGCCAGAAATGGA; //SEQ ID NO: 822TGTGGACCTGCGACGAAGAACGTAAATGCTGCGAAGGTCTGGTTTGCCGTCTGTGGTGCAAACGTATCATCAA//SEQ ID NO: 823 CATG; CTTACATGTTGATGATACGTTTGCACCACAGACGGCAAA;//SEQ ID NO: 824CCAGACCTTCGCAGCATTTACGTTCTTCGTCGCAGGTCCACATCCATTTCTGGCAGTAGGAACCACCACCAGC//SEQ ID NO: 825 GGA;The oligos above were used to form the duplex below:

TGGTTCCGGTGGTGGTGGTTCCTACTGCCAGAAATGGATGTGGACCTGCGACGAAGAACGSEQ ID NO: 826 1---------+---------+---------+---------+---------+---------+ 60    AGGCCACCACCACCAAGGATGACGGTCTTTACCTACACCTGGACGCTGCTTCTTGCSEQ ID NO: 828 G  S  G  G  G  G  S  Y  C  Q  K  W  M  W  T  C  D  E  E  R  -SEQ ID NO: 827 TAAATGCTGCGAAGGTCTGGTTTGCCGTCTGTGGTGCAAACGTATCATCAACATG61 ---------+---------+---------+---------+---------+-----ATTTACGACGCTTCCAGACCAAACGGCAGACACCACGTTTGCATAGTAGTTGTACATTC K  C  C  E  G  L  V  C  R  L  W  C  K  R  I  I  N  M   -

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 25 Fc-L-wGVIA Bacterial Expression

Bacterial expression of Fc-L-wGVIA. The methods to clone and express thepeptibody in bacteria are described in Example 3. The vector used waspAMG21ampR-Fc-Pep and the oligos listed below were used to generate aduplex (see below) for cloning and expression in bacteria of Fc-L-wGVIA.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCTGCAAATCCCCGGGTT; //SEQ ID NO: 829CCTCCTGCTCCCCGACCTCCTACAACTGCTGCCGTTCCTGCAACCCGTACACCAAACGTTGCTACGGT;SEQ ID NO: 830 CTTAACCGTAGCAACGTTTGGTGTACGGGTTGCAGGAA; //SEQ ID NO: 831CGGCAGCAGTTGTAGGAGGTCGGGGAGCAGGAGGAACCCGGGGATTTGCAGGAACCACCACCACCGGA;//SEQ ID NO: 832The oligos above were used to form the duplex below:

TGGTTCCGGTGGTGGTGGTTCCTGCAAATCCCCGGGTTCCTCCTGCTCCCCGACCTCCTASEQ ID NO: 833 1---------+---------+---------+---------+---------+---------+ 60    AGGCCACCACCACCAAGGACGTTTAGGGGCCCAAGGAGGACGAGGGGCTGGAGGATSEQ ID NO: 835 G  S  G  G  G  G  S  C  K  S  P  G  S  S  C  S  P  T  S  Y  -SEQ ID NO: 834 CAACTGCTGCCGTTCCTGCAACCCGTACACCAAACGTTGCTACGGT 61---------+---------+---------+---------+------GTTGACGACGGCAAGGACGTTGGGCATGTGGTTTGCAACGATGCCAATTC N  C  C  R  S  C  N  P  Y  T  K  R  C  Y  G

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 26 Fc-L-ωMVIIA Bacterial Expression

Bacterial expression of Fc-L-ωMVIIA. The methods to clone and expressthe peptibody in bacteria are described in Example 3. The vector usedwas pAMG21ampR-Fc-Pep and the oligos listed below were used to generatea duplex (see below) for cloning and expression in bacteria ofFc-L-ωMVIIA.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCTGCAAAGGTAAA; //SEQ ID NO: 836GGTGCTAAATGCTCCCGTCTGATGTACGACTGCTGCACCGGTTCCTGCCGTTCCGGTAAATGCGGT;//SEQ ID NO: 837 CTTAACCGCATTTACCGGAACGGCAGGAACCGGT; //SEQ ID NO: 838GCAGCAGTCGTACATCAGACGGGAGCATTTAGCACCTTTACCTTTGCAGGAACCACCACCACCGGA;//SEQ ID NO: 839The oligos above were used to form the duplex below:

TGGTTCCGGTGGTGGTGGTTCCTGCAAAGGTAAAGGTGCTAAATGCTCCCGTCTGATGTASEQ ID NO: 840 1---------+---------+---------+---------+---------+---------+ 60    AGGCCACCACCACCAAGGACGTTTCCATTTCCACGATTTACGAGGGCAGACTACATSEQ ID NO: 842 G  S  G  G  G  G  S  C  K  G  K  G  A  K  C  S  R  L  M  Y  -SEQ ID NO: 841 CGACTGCTGCACCGGTTCCTGCCGTTCCGGTAAATGCGGT 61---------+---------+---------+---------+GCTGACGACGTGGCCAAGGACGGCAAGGCCATTTACGCCAATTC D  C  C  T  G  S  C  R  S  G  K  C  G   -

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 27 Fc-L-Ptu1 Bacterial Expression

Bacterial expression of Fc-L-Ptu1. The methods to clone and express thepeptibody in bacteria are described in Example 3. The vector used waspAMG21ampR-Fc-Pep and the oligos listed below were used to generate aduplex (see below) for cloning and expression in bacteria of Fc-L-Ptu1.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCGCTGAAAAAGACTGCATC; //SEQ ID NO: 843GCTCCGGGTGCTCCGTGCTTCGGTACCGACAAACCGTGCTGCAACCCGCGTGCTTGGTGCTCCTCCTACGCTA//SEQ ID NO: 844 ACAAATGCCTG; CTTACAGGCATTTGTTAGCGTAGGAGGAGCACCAAGCACG;//SEQ ID NO: 845CGGGTTGCAGCACGGTTTGTCGGTACCGAAGCACGGAGCACCCGGAGCGATGCAGTCTTTTTCAGCGGAACCA//SEQ ID NO: 846 CCACCACCGGA;The oligos above were used to form the duplex below:

TGGTTCCGGTGGTGGTGGTTCCGCTGAAAAAGACTGCATCGCTCCGGGTGCTCCGTGCTTSEQ ID NO: 847 1---------+---------+---------+---------+---------+---------+  60    AGGCCACCACCACCAAGGCGACTTTTTCTGACGTAGCGAGGCCCACGAGGCACGAASEQ ID NO: 849 G  S  G  G  G  G  S  A  E  K  D  C  I  A  P  G  A  P  C  F -SEQ ID NO: 848CGGTACCGACAAACCGTGCTGCAACCCGCGTGCTTGGTGCTCCTCCTACGCTAACAAATG 61---------+---------+---------+---------+---------+---------+ 120GCCATGGCTGTTTGGCACGACGTTGGGCGCACGAACCACGAGGAGGATGCGATTGTTTAC G  T  K  D  P  C  C  N  P  R  A  W  C  S  S  Y  A  N  K  C - CCTG 121---- GGACATTC  L   -

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 28 Fc-L-ProTx1 Bacterial Expression

Bacterial expression of Fc-L-ProTx1. The methods to clone and expressthe peptibody in bacteria are described in Example 3. The vector usedwas pAMG21ampR-Fc-Pep and the oligos listed below were used to generatea duplex (see below) for cloning and expression in bacteria ofFc-L-ProTx1.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCGAATGCCGTTACTGGCTGG; //SEQ ID NO: 850GTGGTTGCTCCGCTGGTCAGACCTGCTGCAAACACCTGGTTTGCTCCCGTCGTCACGGTTGGTGCGTTTGGGA//SEQ ID NO: 851 CGGTACCTTCTCC;CTTAGGAGAAGGTACCGTCCCAAACGCACCAACCGTGACGA; //SEQ ID NO: 852CGGGAGCAAACCAGGTGTTTGCAGCAGGTCTGACCAGCGGAGCAACCACCCAGCCAGTAACGGCATTCGGAAC//SEQ ID NO: 853 CACCACCACCGGA;The oligos above were used to form the duplex below:

TGGTTCCGGTGGTGGTGGTTCCGAATGCCGTTACTGGCTGGGTGGTTGCTCCGCTGGTCASEQ ID NO: 854 1---------+---------+---------+---------+---------+---------+  60    AGGCCACCACCACCAAGGCTTACGGCAATGACCGACCCACCAACGAGGCGACCAGTSEQ ID NO: 856 G  S  G  G  G  G  S  E  C  R  Y  W  L  G  G  C  S  A  G  Q -SEQ ID NO: 855GACCTGCTGCAAACACCTGGTTTGCTCCCGTCGTCACGGTTGGTGCGTTTGGGACGGTAC 61---------+---------+---------+---------+---------+---------+ 120CTGGACGACGTTTGTGGACCAAACGAGGGCAGCAGTGCCAACCACGCAAACCCTGCCATG T  C  C  K  H  L  V  C  S  R  R  H  G  W  C  V  W  D  G  T - CTTCTCC121 ------- GAAGAGGATTC  F  S   -

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 29 Fc-L-BeKM1 Bacterial Expression

Bacterial expression of Fc-L-BeKM1. The methods to clone and express thepeptibody in bacteria are described in Example 3. The vector used waspAMG21ampR-Fc-Pep and the oligos listed below were used to generate aduplex (see below) for cloning and expression in bacteria of Fc-L-BeKM1.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCCGTCCGACCGACATCAAATG; //SEQ ID NO: 857CTCCGAATCCTACCAGTGCTTCCCGGTTTGCAAATCCCGTTTCGGTAAAACCAACGGTCGTTGCGTTAACGGT//SEQ ID NO: 858 TTCTGCGACTGCTTC;CTTAGAAGCAGTCGCAGAAACCGTTAACGCAACGACCGTTGG; //SEQ ID NO: 859TTTTACCGAAACGGGATTTGCAAACCGGGAAGCACTGGTAGGATTCGGAGCATTTGATGTCGGTCGGACGGGA//SEQ ID NO: 860 ACCACCACCACCGGA;The oligos above were used to form the duplex below:

TGGTTCCGGTGGTGGTGGTTCCCGTCCGACCGACATCAAATGCTCCGAATCCTACCAGTGSEQ ID NO: 861 1---------+---------+---------+---------+---------+---------+  60    AGGCCACCACCACCAAGGGCAGGCTGGCTGTAGTTTACGAGGCTTAGGATGGTCACSEQ ID NO: 863 G  S  G  G  G  G  S  R  P  T  D  I  K  C  S  E  S  Y  Q  C -SEQ ID NO: 862CTTCCCGGTTTGCAAATCCCGTTTCGGTAAAACCAACGGTCGTTGCGTTAACGGTTTCTG 61---------+---------+---------+---------+---------+---------+ 120GAAGGGCCAAACGTTTAGGGCAAAGCCATTTTGGTTGCCAGCAACGCAATTGCCAAAGAC F  P  V  C  K  S  R  F  G  K  T  N  G  R  C  V  N  G  F  C - CGACTGCTTC121 ---------+ GCTGACGAAGATTC  D  C  F   -

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 30 Fc-L-CTX Bacterial Expression

Bacterial expression of Fc-L-CTX. The methods to clone and express thepeptibody in bacteria are described in Example 3. The vector used waspAMG21ampR-Fc-Pep and the oligos listed below were used to generate aduplex (see below) for cloning and expression in bacteria of Fc-L-CTX.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCATGTGCATGCCGTGCTTCAC; //SEQ ID NO: 864CACCGACCACCAGATGGCTCGTAAATGCGACGACTGCTGCGGTGGTAAAGGTCGTGGTAAATGCTACGGTCCG//SEQ ID NO: 865 CAGTGCCTGTGCCGT;CTTAACGGCACAGGCACTGCGGACCGTAGCATTTACCACGAC; //SEQ ID NO: 866CTTTACCACCGCAGCAGTCGTCGCATTTACGAGCCATCTGGTGGTCGGTGGTGAAGCACGGCATGCACATGGA//SEQ ID NO: 867 ACCACCACCACCGGA;The oligos above were used to form the duplex below:

TGGTTCCGGTGGTGGTGGTTCCATGTGCATGCCGTGCTTCACCACCGACCACCAGATGGCSEQ ID NO: 868 1---------+---------+---------+---------+---------+---------+  60    AGGCCACCACCACCAAGGTACACGTACGGCACGAAGTGGTGGCTGGTGGTCTACCGSEQ ID NO: 870 G  S  G  G  G  G  S  M  C  M  P  C  F  T  T  D  H  Q  M  A -SEQ ID NO: 869TCGTAAATGCGACGACTGCTGCGGTGGTAAAGGTCGTGGTAAATGCTACGGTCCGCAGTG 61---------+---------+---------+---------+---------+---------+ 120AGCATTTACGCTGCTGACGACGCCACCATTTCCAGCACCATTTACGATGCCAGGCGTCAC R  K  C  D  D  C  C  G  G  K  G  R  G  K  C  Y  G  P  Q  C - CCTGTGCCGT121 ---------+ GGACACGGCACCAC  L  C  R   -

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 31 N-Terminally PEGylated-Des-Arg1-ShK

Peptide Synthesis of reduced Des-Arg1-ShK. Des-Arg1-ShK, having thesequence

(Peptide 1, SEQ ID NO: 92) SCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTCwas synthesized in a stepwise manner on a Symphony™ multi-peptidesynthesizer by solid-phase peptide synthesis (SPPS) using2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU)/N-methyl morpholine (NMM)/N,N-dimethyl-formamide (DMF) couplingchemistry at 0.1 mmol equivalent resin scale on Tentagel™-S PHBFmoc-Cys(Trt)-resin. N-alpha-(9-fluorenylmethyloxycarbonyl)- andside-chain protected amino acids were purchased from Midwest BiotechIncorporated. Fmoc-Cys(Trt)-Tentagel™ resin was purchased from Fluka.The following side-chain protection strategy was employed: Asp(O^(t)Bu),Arg(Pbf), Cys(Trt), Gln(Trt), His(Trt), Lys(N^(ε)-Boc), Ser(O^(t)Bu),Thr(O^(t)Bu) and Tyr(O^(t)Bu). Two Oxazolidine dipeptides,Fmoc-Gly-Thr(^(ψMe, Me)Pro)-OH and Fmoc-Leu-Ser(^(ψMe,Me)Pro)-OH, wereused in the chain assembly and were obtained from NovaBiochem and usedin the synthesis of the sequence. The protected amino acid derivatives(20 mmol) were dissolved in 100 ml 20% dimethyl sulfoxide (DMSO) in DMF(v/v). Protected amino acids were activated with 20 mM HBTU, 400 mM NMMin 20% DMSO in DMF, and coupling were carried out using two treatmentswith 0.5 mmol protected amino acid, 0.5 mmol HBTU, 1 mmol NMM in 20%DMF/DMSO for 25 minutes and then 40 minutes. Fmoc deprotection reactionswere carried out with two treatments using a 20% piperidine in DMF (v/v)solution for 10 minutes and then 15 minutes. Following synthesis, theresin was then drained, and washed with DCM, DMF, DCM, and then dried invacuo. The peptide-resin was deprotected and released from the resin bytreatment with a TFA/EDT/TIS/H₂O (92.5:2.5:2.5:2.5 (v/v)) solution atroom temperature for 1 hour. The volatiles were then removed with astream of nitrogen gas, the crude peptide precipitated twice with colddiethyl ether and collected by centrifugation. The crude peptide wasthen analyzed on a Waters 2795 analytical RP-HPLC system using a lineargradient (0-60% buffer B in 12 minutes, A: 0.1% TFA in water, B: 0.1%TFA in acetonitrile) on a Jupiter 4 μm Proteo™ 90 Å column. A PE-Sciex™API Electro-spray mass spectrometer was used to confirm correct peptideproduct mass. Crude peptide was obtained in 143 mg yield atapproximately 70% pure based as estimated by analytical RP-HPLCanalysis. Reduced Des-Arg1-ShK (Peptide 1) Retention time (Rt)=5.31minutes, calculated molecular weight=3904.6917 Da (average);Experimental observed molecular weight 3907.0 Da.

Folding of Des-Arg1-ShK (Disulphide bond formation). Following TFAcleavage and peptide precipitation, reduced Des-Arg1-ShK was thenair-oxidized to give the folded peptide. The crude cleaved peptide wasextracted using 20% AcOH in water (v/v) and then diluted with water to aconcentration of approximately 0.15 mg reduced Des-Arg1-ShK per mL, thepH adjusted to about 8.0 using NH₄OH (28-30%), and gently stirred atroom temperature for 36 hours. Folding process was monitored by LC-MSanalysis. Following this, folded Des-Arg1-ShK peptide was purified usingreversed phase HPLC using a 1″ Luna 5 μm C18 100 Å Proteo™ column with alinear gradient 0-40% buffer B in 120 min (A=0.1% TFA in water, B=0.1%TFA in acetonitrile). Folded Des-Arg1-ShK crude peptide eluted earlier(when compared to the elution time in its reduced form) at approximately25% buffer B. Folded Des-Arg1-ShK (Peptide 2) was obtained in 23.2 mgyield in >97% purity as estimated by analytical RP-HPLC analysis (FIG.20). Calculated molecular weight=3895.7693 Da (monoisotopic),experimental observed molecular weight=3896.5 Da (analyzed on a WatersLCT Premier Micromass MS Technologies). Des-Arg1-ShK disulfideconnectivity was C1-C6, C2-C4, C3-C5.

N-terminal PEGylation of Folded Des-Arg1-ShK. Folded Des-Arg1-ShK,(Peptide 2) was dissolved in water at 1 mg/ml concentration. A 2 MMeO-PEG-Aldehyde, CH₃O—[CH₂CH₂O]_(n)—CH₂CH₂CHO (average molecular weight20 kDa), solution in 50 mM NaOAc, pH 4.5, and a separate 1 M solution ofNaCNBH₃ were freshly prepared. The peptide solution was then added tothe MeO-PEG-Aldehyde containing solution and was followed by theaddition of the NaCNBH₃ solution. The reaction stoichiometry waspeptide:PEG:NaCNBH3 (1:2:0.02), respectively. The reaction was left for48 hours, and was analyzed on an Agilent 1100 RP-HPLC system usingZorbax™ 300SB-C8 5 μm column at 40° C. with a linear gradient (6-60% Bin 16 minutes, A: 0.1% TFA in water, B: 0.1% TFA/90% ACN in water).Mono-pegylated folded Des-Arg1-ShK constituted approximately 58% of thecrude product by analytical RP-HPLC. Mono Pegylated Des-Arg1-ShK wasthen isolated using a HiTrap™ 5 ml SP HP cation exchange column on AKTAFPLC system at 4° C. at 1 mL/min using a gradient of 0-50% B in 25column volumes (Buffers: A=20 mM sodium acetate pH 4.0, B=1 M NaCl, 20mM sodium acetate, pH 4.0). The fractions were analyzed using a 4-20tris-Gly SDS-PAGE gel and RP-HPLC (as described for the crude). SDS-PAGEgels were run for 1.5 hours at 125 V, 35 mA, 5 W. Pooled product wasthen dialyzed at 4° C. in 3 changes of 1 L of A4S buffer (10 mM NaOAc,5% sorbitol, pH 4.0). The dialyzed product was then concentrated in 10 KmicroCentrifuge filter to 2 mL volume and sterile-filtered using 0.2 μMsyringe filter to give the final product. N-TerminallyPEGylated-Des-Arg1-ShK (Peptide 3) was isolated in 1.7 mg yield with 85%purity as estimated by analytical RP-HPLC analysis (FIG. 23).

The N-Terminally PEGylated-Des-Arg1-ShK, also referred to as“PEG-ShK[2-35]”, was active in blocking human Kv1.3 (FIG. 38A and FIG.38B) as determined by patch clamp electrophysiology (Example 36).

Example 32 N-Terminally PEGylated ShK

The experimental procedures of this working example correspond to theresults shown in FIG. 17.

Peptide Synthesis of reduced ShK. ShK, having the amino acid sequence

(Peptide 4, SEQ ID NO: 5) RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTCwas synthesized in a stepwise manner on a Symphony™ multi-peptidesynthesizer by solid-phase peptide synthesis (SPPS) using2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU)/N-methyl morpholine (NMM)/N,N-dimethyl-formamide (DMF) couplingchemistry at 0.1 mmol equivalent resin scale on Tentagel™-S PHBFmoc-Cys(Trt)-resin. N-alpha-9-fluorenylmethyloxycarbonyl) andside-chain protected amino acids were purchased from Midwest BiotechIncorporated. Fmoc-Cys(Trt)-Tentagel™ resin was purchased from Fluka.The following side-chain protection strategy was employed: Asp(O^(t)Bu),Arg(Pbf), Cys(Trt), Gln(Trt), His(Trt), Lys(N^(ε)-Boc), Ser(O^(t)Bu),Thr(O^(t)Bu) and Tyr(O^(t)Bu). Two Oxazolidine dipeptides,Fmoc-Gly-Thr(^(ψMe, Me)Pro)-OH and Fmoc-Leu-Ser(^(ψMe, Me)Pro)-OH, wereused in the chain assembly and were obtained from NovaBiochem and usedin the synthesis of the sequence. The protected amino acid derivatives(20 mmol) were dissolved in 100 ml 20% dimethyl sulfoxide (DMSO) in DMF(v/v). Protected amino acids were activated with 200 mM HBTU, 400 mM NMMin 20% DMSO in DMF, and coupling were carried out using two treatmentswith 0.5 mmol protected amino acid, 0.5 mmol HBTU, 1 mmol NMM in20%DMF/DMSO for 25 minutes and then 40 minutes. Fmoc deprotections werecarried out with two treatments using a 20% piperidine in DMF (v/v)solution for 10 minutes and then 15 minutes. Following synthesis, theresin was then drained, and washed with DCM, DMF, DCM, and then dried invacuo. The peptide-resin was deprotected and released from the resin bytreatment with a TFA/EDT/TIS/H₂O (92.5:2.5:2.5:2.5 (v/v)) solution atroom temperature for 1 hour. The volatiles were then removed with astream of nitrogen gas, the crude peptide precipitated twice with colddiethyl ether and collected by centrifugation. The crude peptide wasthen analyzed on a Waters 2795 analytical RP-HPLC system using a lineargradient (0-60% buffer B in 12 minutes, A: 0.1% TFA in water, B: 0.1%TFA in acetonitrile) on a Jupiter 4 μm Proteo™ 90 Å column. A PE-SciexAPI Electro-spray mass spectrometer was used to confirm correct peptideproduct mass. Crude peptide was approximately was obtained 170 mg yieldat about 45% purity as estimated by analytical RP-HPLC analysis. ReducedShK (Peptide 4) Retention time (Rt)=5.054 minutes, calculated molecularweight=4060.8793 Da (average); experimental observed molecularweight=4063.0 Da.

Folding of ShK (Disulphide bond formation). Following TFA cleavage andpeptide precipitation, reduced ShK was then air oxidized to give thefolded peptide. The crude cleaved peptide was extracted using 20% AcOHin water (v/v) and then diluted with water to a concentration ofapproximately 0.15 mg reduced ShK per mL, the pH adjusted to about 8.0using NH₄OH (28-30%), and gently stirred at room temperature for 36hours. Folding process was monitored by LC-MS analysis. Following this,folded ShK peptide was purified by reversed phase HPLC using a 1″ Luna 5μm C18 100 Å Proteo™ column with a linear gradient 0-40% buffer B in 120min (A=0.1% TFA in water, B=0.1% TFA in acetonitrile). Folded ShK crudepeptide eluted earlier (when compared to the elution time in its reducedform) at approximately 25% buffer B. Folded ShK (Peptide 5) was obtainedin 25.5 mg yield in >97% purity as estimated by analytical RP-HPLCanalysis. See FIG. 60. Calculated molecular weight=4051.8764 Da(monoisotopic); experimental observed molecular weight=4052.5 Da(analyzed on Waters LCT Premier Micromass MS Technologies). ShKdisulfide connectivity was C1-C6, C2-C4, and C3-C5.

N-terminal PEGylation of Folded ShK. Folded ShK, having the amino acidsequence

(SEQ ID NO: 5) RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTCcan be dissolved in water at 1 mg/ml concentration. A 2 MMeO-PEG-Aldehyde, CH₃O—CH₂CH₂O]_(n)—CH₂CH₂CHO (average molecular weight20 kDa), solution in 50 mM NaOAc, pH 4.5 and a separate 1 M solution ofNaCNBH₃ can be freshly prepared. The peptide solution can be then addedto the MeO-PEG-Aldehyde containing solution and can be followed by theaddition of the NaCNBH₃ solution. The reaction stoichiometry can bepeptide:PEG:NaCNBH3 (1:2:0.02), respectively. The reaction can be leftfor 48 hours, and can be analyzed on an Agilent™ 1100 RP-HPLC systemusing Zorbax™ 300SB-C8 5 μm column at 40° C. with a linear gradient(6-60% B in 16 minutes, A: 0.1% TFA in water, B: 0.1% TFA/90% ACN inwater). Mono-pegylated Shk (Peptide 6) can be then isolated using aHiTrap™ 5 mL SP HP cation exchange column on AKTA FPLC system at 4° C at1 mL/min using a gradient of 0-50% B in 25 column volumes (Buffers: A=20mM sodium acetate pH 4.0, B=1 M NaCl, 20 mM sodium acetate, pH 4.0). Thefractions can be analyzed using a 4-20 tris-Gly SDS-PAGE gel andRP-HPLC. SDS-PAGE gels can be run for 1.5 hours at 125 V, 35 mA, 5 W.Pooled product can be then dialyzed at 4° C. in 3 changes of 1 L of A4Sbuffer (10 mM sodium acetate, 5% sorbitol, pH 4.0). The dialyzed productcan be then concentrated in 10 K microCentrifuge filter to 2 mL volumeand sterile-filtered using 0.2 μM syringe filter to give the finalproduct.

Example 33 N-Terminally PEGylated ShK by Oxime Formation

Peptide Synthesis of reduced ShK. ShK, having the sequence

(SEQ ID NO: 5) RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTCcan be synthesized in a stepwise manner on a Symphony™ multi-peptidesynthesizer by solid-phase peptide synthesis (SPPS) using2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU)/N-methyl morpholine (NMM)/N,N-dimethyl-formamide (DMF) couplingchemistry at 0.1 mmol equivalent resin scale on Tentagel™-S PHBFmoc-Cys(Trt)-resin. N-alpha-(9-fluorenylmethyloxycarbonyl)- andside-chain protected amino acids can be purchased from Midwest BiotechIncorporated. Fmoc-Cys(Trt)-Tentagel™ resin can be purchased from Fluka.The following side-chain protection strategy can be employed:Asp(O^(t)Bu), Arg(Pbf), Cys(Trt), Gln(Trt), His(Trt), Lys(N^(ε)-Boc),Ser(O^(t)Bu), Thr(O^(t)Bu) and Tyr(O^(t)Bu). Two Oxazolidine dipeptides,Fmoc-Gly-Thr(Ψ^(Me, Me)Pro)-OH and Fmoc-Leu-Ser(ψ^(Me, Me)Pro)-OH, canbe used in the chain assembly and can be obtained from NovaBiochem andused in the synthesis of the sequence. The protected amino acidderivatives (20 mmol) can be dissolved in 100 ml 20% dimethyl sulfoxide(DMSO) in DMF (v/v). Protected amino acids can be activated with 200 mMHBTU, 400 mM NMM in 20% DMSO in DMF, and coupling can be carried outusing two treatments with 0.5 mmol protected amino acid, 0.5 mmol HBTU,1 mmol NMM in 20% DMF/DMSO for 25 minutes and then 40 minutes. Fmocdeprotection reactions can be carried out with two treatments using a20% piperidine in DMF (v/v) solution for 10 minutes and then 15 minutes.Following the chain-assembly of the Shk peptide, Boc-amionooxyaceticacid (1.2 equiv) can be coupled at the N-terminus using 0.5 M HBTU inDMF with 4 equiv collidine for 5 minutes. Following synthesis, the resincan be then drained, and washed with DCM, DMF, DCM, and then dried invacuo. The peptide-resin can be deprotected and released from the resinby treatment with a TFA/amionooxyacetic acid/TIS/EDT/H2O(90:2.5:2.5:2.5:2.5) solution at room temperature for 1 hour. Thevolatiles can be then removed with a stream of nitrogen gas, the crudepeptide precipitated twice with cold diethyl ether and collected bycentrifugation. The aminooxy-Shk peptide (Peptide 7) can be thenanalyzed on a Waters 2795 analytical RP-HPLC system using a lineargradient (0-60% buffer B in 12 minutes, A: 0.1% TFA in water alsocontaining 0.1% aminooxyacetic acid, B: 0.1% TFA in acetonitrile) on aJupiter 4 μm Proteo™ 90 Å column.

Reversed-Phase HPLC Purification. Preparative Reversed-phasehigh-performance liquid chromatography can be performed on C18, 5 μm,2.2 cm×25 cm) column. Chromatographic separations can be achieved usinglinear gradients of buffer B in A (A=0.1% aqueous TFA; B=90% aq. ACNcontaining 0.09% TFA and 0.1% aminooxyacetic acid), typically 5-95% over90 minutes at 15 mL/min. Preparative HPLC fractions can be characterizedby ESMS and photodiode array (PDA) HPLC, combined and lyophilized.

N-Terminal PEGylation of Shk by Oxime Formation. Lyophilizedaminooxy-Shk (Peptide 7) can be dissolved in 50% HPLC buffer A/B (5mg/mL) and added to a two-fold molar excess of MeO-PEG-Aldehyde,CH₃O—[CH₂CH₂O]_(n)—CH₂CH₂CHO (average molecular weight 20 kDa). Thereaction can be left for 24 hours, and can be analyzed on an Agilent™1100 RP-HPLC system using Zorbax™ 300SB-C8 5 μm column at 40° C. with alinear gradient (6-60% B in 16 minutes, A: 0.1% TFA in water, B: 0.1%TFA/90% ACN in water). Mono-pegylated reduced Shk constitutedapproximately 58% of the crude product by analytical RP-HPLC. MonoPEGylated (oximated) Shk (Peptide 8) can be then isolated using aHiTrap™ 5 mL SP HP cation exchange column on AKTA FPLC system at 4° C.at 1 mL/min using a gradient of 0-50% B in 25 column volumes (Buffers:A=20 mM sodium acetate pH 4.0, B=1 M NaCl, 20 mM sodium acetate, pH4.0). The fractions can be analyzed using a 4-20 tris-Gly SDS-PAGE geland RP-HPLC. SDS-PAGE gels can be run for 1.5 hours at 125 V, 35 mA, 5W. Pooled product can be then dialyzed at 4° C. in 3 changes of 1 L ofA4S buffer (10 mM NaOAc, 5% sorbitol, pH 4.0). The dialyzed product canbe then concentrated in 10 K microCentrifuge filter to 2 mL volume andsterile-filtered using 0.2 μM syringe filter to give the final product.

Folding of ShK (Disulphide bond formation). The mono-PEGylated(oximated) Shk can be dissolved in 20% AcOH in water (v/v) and can bethen diluted with water to a concentration of approximately 0.15 mgpeptide mL, the pH adjusted to about 8.0 using NH₄OH (28-30%), andgently stirred at room temperature for 36 hours. Folding process can bemonitored by LC-MS analysis. Following this, folded mono-PEGylated(oximated) Shk (Peptide 9) can be purified using by reversed phase HPLCusing a 1″ Luna 5 μm C18 100 Å Proteo™ column with a linear gradient0-40% buffer B in 120 min (A=0.1% TFA in water, B=0.1% TFA inacetonitrile). Mono-PEGylated (oximated) ShK disulfide connectivity canbe C1-C6, C2-C4, and C3-C5.

Example 34 N-Terminally PEGylated ShK (Amidation)

The experimental procedures of this working example correspond to theresults shown in FIG. 18.

N-Terminal PEGylation of Shk by Amide Formation. A 10 mg/mL solution offolded Shk (Peptide 5), in 100 mM Bicine pH 8.0, can be added to solidsuccinimidyl ester of 20 kDa PEG propionic acid (mPEG-SPA;CH₃O—[CH₂CH₂O]_(n)—CH₂CH₂CO—NHS) at room temperature using a 1.5 molarexcess of the mPEG-SPA to Shk. After one hour with gentle stirring, themixture can be diluted to 2 mg/mL with water, and the pH can be adjustedto 4.0 with dilute HCl. The extent of mono-pegylated Shk (Peptide 10),some di-PEGylated Shk or tri-PEGylated Shk, unmodified Shk andsuccinimidyl ester hydrolysis can be determined by SEC HPLC using aSuperdex™ 75 HR 10/30 column (Amersham) eluted with 0.05 M NaH₂PO₄, 0.05M Na₂HPO₄, 0.15 M NaCl, 0.01 M NaN₃, pH 6.8, at 1 mL/min. The fractionscan be analyzed using a 4-20 tris-Gly SDS-PAGE gel and RP-HPLC. SDS-PAGEgels can be run for 1.5 hours at 125 V, 35 mA, 5 W. Pooled product canbe then dialyzed at 4° C. in 3 changes of 1 L of A4S buffer (10 mMNaOAc, 5% sorbitol, pH 4.0). The dialyzed N-terminally PEGylated(amidated) ShK (Peptide 10) can be then concentrated in 10 KmicroCentrifuge filter to 2 mL volume and sterile-filtered using 0.2 μMsyringe filter to give the final product.

Example 35 Fc-L-SmIIIA

Fc-SmIIIA expression vector. A 104 bp BamHI-NotI fragment containingpartial linker sequence and SmIIIA peptide encoded with human highfrequency codons was assembled by PCR with overlapping primers 3654-50and 3654-51 and cloned into to the 7.1 kb NotI-BamHI back bone togenerate pcDNA3.1(+)CMVi-hFc-SmIIIA as described in Example 1.

   BamHI 5′GGATCCGGAGGAGGAGGAAGCTGCTGCAACGGCCGCCGCGGCTGCAGCAGCCGCTGG//SEQ ID NO: 872                        C  C  N  G  R  R  G  C  S  S  R  WSEQ ID NO: 873 TGCCGCGACCACAGCCGCTGCTGCTGAGCGGCCGC3′C  R  D  H  S  R  C  C        NotI Forward 5′-3′GGAGGAGGATCCGGAGGAGGAGGAAGCTGCTGCAACGGCCGCCGCGGCTGCAGCAGC CGC//SEQ ID NO: 874 Reverse 5′-3′ATTATTGCGGCCGCTCAGCAGCAGCGGCTGTGGTCGCGGCACCAGCGGCTGCTGCAG CCGCSEQ ID NO: 875The sequences of the BamHI to NotI fragments in the final constructswere verified by sequencing.

Transient expression of Fc-L-SmIIIa. 7.5 ug of the toxin peptide Fcfusion construct pcDNA3.1(+)CMVi-hFc-SmIIIA were transfected into 293-Tcells in 10 cm tissue culture plate with FuGENE 6 as transfectionreagent. Culture medium was replaced with serum-free medium at 24 hourspost-transfection and the conditioned medium was harvested at day 5post-transfection. Transient expression of Fc-SmIIIA from 293-T cellswas analyzed by Western blot probed with anti-hFc antibody (FIG. 25A andFIG. 25B). Single band of expressed protein with estimated MW was shownin both reduced and non-reduced samples. Transient expression level ofFc-SmIIIA was further determined to be 73.4 μg/ml according to ELISA.

Example 36 Electrophysiology Experiments

Cell Culture. Stable cell line expressing human Kv1.3 channel waslicensed from Biofocus. Cells were kept at 37° C. in 5% CO₂ environment.Culture medium contains DMEM with GlutaMax™ (Invitrogen), 1×non-essential amino acid, 10% fetal bovine serum and 500 μg/mLgeneticin. Cells were plated and grown at low confluence on 35 mmculture dishes for at least 24 hours prior to electrophysiologyexperiments.

Electrophysiology Recording by Patch Clamping. Whole-cell currents wererecorded from single cells by using tight seal configuration of thepatch-clamp technique. A 35 mm culture dish was transferred to therecording stage after rinsing and replacing the culture medium withrecording buffer containing 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl₂, 10 mMHEPES, and 5 mM Glucose. pH was adjusted to 7.4 with NaOH and theosmolarity was set at 300 mOsm. Cells were perfused continuously withthe recording buffer via one of the glass capillaries arranged inparallel and attached to a motorized rod, which places the glasscapillary directly on top of the cell being recorded. Recording pipettesolution contained 90 mM K-gluconate, 20 mM KF, 10 mM NaCl, 1 mMMgCl₂-6H₂O, 10 mM EGTA, 5 mM K₂-ATP, and 10 mM HEPES. The pH for theinternal solution was adjusted to 7.4 with KOH and the osmolarity wasset at 280 mOsm. Experiments were performed at room temperature (20-22°C.) and recorded using Multiclamp™ 700A amplifier (Molecular DevicesInc.). Pipette resistances were typically 2-3 MΩ.

Protein Toxin potency determination on Kv1.3 current: HEK293 cellsstably expressing human Kv1.3 channel were voltage clamped at −80 mVholding potential. Outward Kv1.3 currents were activated by giving 200msec long depolarizing steps to +30 mV from the holding potential of −80mV and filtered at 3 kHz. Each depolarizing step was separated from thesubsequent one with a 10 s interval. Analogue signals were digitized byDigidata™ 1322A digitizer (Molecular Devices) and subsequently stored oncomputer disk for offline analyses using Clampfit™ 9 (Molecular DevicesInc.). In all studies, stable baseline Kv1.3 current amplitudes wereestablished for 4 minutes before starting the perfusion of the proteintoxin at incremental concentrations. A steady state block was alwaysachieved before starting the perfusion of the subsequent concentrationof the protein toxin.

Data analysis. Percent of control (POC) is calculated based on thefollowing equation: (Kv1.3 current after protein toxin addition/Kv1.3current in control)*100. At least 5 concentrations of the protein toxin(e.g. 0.003, 0.01, 0.03, 0.1, 0.3, 100 nM) were used to calculate theIC₅₀ value. IC₅₀ values and curve fits were estimated using the fourparameter logistic fit of XLfit software (Microsoft Corp.). IC₅₀ valuesare presented as mean value±s.e.m. (standard error of the mean).

Drug preparations. Protein toxins (typically 10-100 μM) were dissolvedin distilled water and kept frozen at −80° C. Serial dilutions of thestock protein toxins were mixed into the recording buffer containing0.1% bovine serum albumin (BSA) and subsequently transferred to glassperfusion reservoirs. Electronic pinch valves controlled the flow of theprotein toxin from the reservoirs onto the cell being recorded.

Example 37 Immunobiology and Channel Binding

Inhibition of T cell cytokine production following PMA and anti-CD3antibody stimulation of PBMCs. PBMC's were previously isolated fromnormal human donor Leukophoresis packs, purified by density gradientcentrifugation (Ficoll Hypaque), cryopreserved in CPZ CryopreservationMedium Complete (INCELL, MCPZF-100 plus 10% DMSO final). PBMC's werethawed (95% viability), washed, and seeded at 2×10⁵ cells per well inculture medium (RPMI medium 1640; GIBCO) supplemented with 10% fetalcalf serum, 100 U/ml penicillin, 100 mg/ml streptomycin 2 mML-glutamine, 100 uM non-essential amino acids, and 20 uM 2-ME) in96-well flat-bottom tissue culture plates. Cells were pre-incubated withserially diluted (100 nM-0.001 nM final) ShK[1-35], Fc-L10-ShK[1-35] orfc control for 90 min before stimulating for 48 hr with PMA/anti-CD3 (1ng/ml and 50 ng/ml, respectively) in a final assay volume of 200 ul.Analysis of the assay samples was performed using the Meso ScaleDiscovery (MSD) SECTOR™ Imager 6000 (Meso Scale Discovery, Gaithersbury,Md.) to measure the IL-2 and IFNg protein levels by utilizingelectrochemiluminescence (ECL). The conditioned medium (50 ul) was addedto the MSD Multi-spot 96-well plates (each well containing three captureantibodies; IL-2, TNF, IFNγ). The plates were sealed, wrapped in tinfoil, and incubated at room temperature on a plate shaker for 2 hr. Thewells were washed 1× with 200 ul PBST (BIOTEK, Elx405 Auto PlateWasher). For each well, 20 ul of Ruthenium-labeled detection antibodies(1 ug/ml final in Antibody Dilution Buffer; IL-1, TNF, IFNγ) and 130 ulof 2×MSD Read Buffer added, final volume 150 ul. The plates were sealed,wrapped in tin foil, and incubated at room temperature on a plate shakerfor 1 hr. The plates were then read on the SECTOR™ Imager 6000. FIGS.35A & 35B shows the CHO-derived Fc-L10-ShK[1-35] peptibody potentlyinhibits IL-2 and IFNg production from T cells in a dose-dependentmanner. Compared to native ShK[1-35] peptide, the peptibody produces agreater extent of inhibition (POC=Percent Of Control of response in theabsence of inhibitor).

Inhibition of T cell cytokine production following Anti-CD3 andanti-CD28 antibody stimulation of PBMCs. PBMCs were previously isolatedfrom normal human donor Leukopheresis packs, purified by densitygradient centrifugation (Ficoll Hypaque), and cryopreserved using INCELLFreezing Medium. PBMCs were thawed (95% viability), washed, and seeded(in RPMI complete medium containing serum replacement, PSG) at 2×10⁵cells per well into 96-well flat bottom plates. Cells were pre-incubatedwith serially diluted (100 nM-0.003 nM final) ShK[1-35],Fc-L10-ShK[1-35], or Fc control for 1 hour before the addition of aCD3and aCD28 (2.5 ng/mL and 100 ng/mL respectively) in a final assay volumeof 200 mL. Supernatants were collected after 48 hours, and analyzedusing the Meso Scale Discovery (MSD) SECTOR™ Imager 6000 (Meso ScaleDiscovery, Gaithersbury, Md.) to measure the IL-2 and IFNg proteinlevels by utilizing electrochemiluminescence (ECL). 20 mL of supernatantwas added to the MSD multi-spot 96-well plates (each well containingIL-2, TNFa, and IFNg capture antibodies). The plates were sealed andincubated at room temperature on a plate shaker for 1 hour. Then 20 mLof Ruthenium-labeled detection antibodies (1 ug/ml final of IL-2, TNFα,and IFNγ in Antibody Dilution Buffer) and 110 mL of 2×MSD Read Bufferwere added. The plates were sealed, covered with tin foil, and incubatedat room temperature on a plate shaker for 1 hour. The plates were thenread on the SECTOR™ Imager 6000. FIGS. 37A & 37B shows the CHO-derivedFc-L10-ShK[1-35] peptibody potently inhibits IL-2 and IFNg productionfrom T cells in a dose-dependent manner. Compared to native ShK[1-35]peptide which shows only partial inhibtion, the peptibody producesnearly complete inhibitiono of the inflammatory cytokine response.(POC=Percent Of Control of response in the absence of inhibitor).

Inhibition of T cell proliferation following anti-CD3 and anti-CD28antibody stimulation of PBMCs. PBMC's were previously isolated fromnormal human donor Leukophoresis packs, purified by density gradientcentrifugation (Ficoll Hypaque), cryopreserved in CPZ CryopreservationMedium Complete (INCELL, MCPZF-100 plus 10% DMSO final). PBMC's werethawed (95% viability), washed, and seeded at 2×10⁵ cells per well inculture medium (RPMI medium 1640; GIBCO) supplemented with 10% fetalcalf serum, 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mML-glutamine, 100 μM non-essential amino acids, and 20 μM 2-ME) in96-well flat-bottom tissue culture plates. Cells were pre-incubated witheither anti-human CD32 (FcyRII) blocking antibody (per manufacturersinstructions EASY SEP Human Biotin Selection Kit #18553, StemCellTechnologies Vancouver, BC) or Fc-L10-ShK (100 nM-0.001 nM final) for 45min. Fc-L10-ShK (100 nM-0.001 nM final) was then added to the cellscontaining anti-human CD32 blocking antibody while medium was added tothe cells containing Fc-L10-ShK. Both sets were incubated for anadditional 45 min before stimulating for 48 hr with aCD3/aCD28 (0.2ng/ml and 100 ng/ml, respectively). Final assay volume was 200 ul.[3H]TdR (1 uCi per well) was added and the plates were incubated for anadditional 16 hrs. Cells were then harvested onto glass fiber filtersand radioactivity was measured in a B-scintillation counter. FIGS. 36A &36B shows the CHO-derived Fc-L10-ShK[1-35] peptibody potently inhibitsproliferation of T cells in a dose-dependent manner. Pre-blocking withthe anti-CD32 (FcR) blocking antibody has little effect on thepeptibodies ability to inhibit T cell proliferation suggesting Kv1.3inhibition and not FcR binding is the mechanism for the inhibitionobserved (POC=Percent Of Control of response in the absence ofinhibitor).

Immunohistochemistry analysis of Fc-L10-ShK[1-35] binding to HEK 293cells overexpressing human Kv1.3. HEK 293 cells overexpressing humanKv1.3 (HEK Kv1.3) were obtained from BioFocus plc (Cambridge, UK) andmaintained per manufacturer's recommendation. The parental HEK 293 cellline was used as a control. Cells were plated on Poly-D-Lysine 24 wellplates (#35-4414; Becton-Dickinson, Bedford, Mass.) and allowed to growto approximately 70% confluence. HEK KV1.3 were plated at 0.5×10e5cells/well in 1 ml/well of medium. HEK 293 cells were plated at adensity of 1.5×10e5 cells/well in 1 ml/well of medium. Before staining,cells were fixed with formalin (Sigma HT50-1-1 Formalin solution,diluted 1:1 with PBS/0.5% BSA before use) by removing cell growthmedium, adding 0.2 ml/well formalin solution and incubating at roomtemperature for ten minutes. Cells were stained by incubating with 0.2ml/well of 5 ug/ml Fc-L10-ShK[1-35] in PBS/BSA for 30′ at roomtemperature. Fc-L10-ShK[1-35] was aspirated and then the cells werewashed one time with PBS/0.5% BSA. Detection antibody (Goat F(ab)2anti-human IgG-phycoerythrin; Southern Biotech Associates, Birmingham,Ala.) was added to the wells at 5 ug/ml in PBS/0.5% BSA and incubatedfor 30′ at room temperature. Wash cells once with PBS/0.5% BSA andexamine using confocal microscopy (LSM 510 Meta Confocal Microscope;Carl Zeiss AG, Germany). FIG. 33B shows the Fc-L10-ShK[1-35] peptibodyretains binds to Kv1.3 overexpressing HEK 293 cells but shows littlebinding to untransfected cells (FIG. 33A) indicating theFc-L10-ShK[1-35] peptibody can be used as a reagent to detect cellsoverexpressing the Kv1.3 channel. In disease settings where activated Teffector memory cells have been reported to overproduce Kv1.3, thisreagent can find utility in both targeting these cells and in theirdetection.

An ELISA assay demonstrating Fc-L10-ShK[1-35] binding to fixed HEK 293cells overexpressing Kv1.3. FIG. 34A shows a dose-dependent increase inthe peptibody binding to fixed cells that overexpress Kv1.3,demonstrating that the peptibody shows high affinity binding to itstarget and the utility of the Fc-L10-ShK[1-35] molecule in detection ofcells expressing the channel. Antigen specific T cells that causedisease in patients with multiple sclerosis have been shown tooverexpress Kv1.3 by whole cell patch clamp electrophysiology,—alaborius approach. Our peptibody reagent can be a useful and convenienttool for monitoring Kv1.3 channel expression in patients and haveutility in diagnostic applications. The procedure shown in FIG. 34A andFIG. 34B follows.

FIG. 34A. A whole cell immunoassay was performed to show binding ofintact Fc-L10-ShK[1-35] to Kv1.3 transfected HEK 293 cells (BioFocusplc, Cambridge, UK). Parent HEK 293 cells or HEK Kv1.3 cells were platedat 3×10e4 cells/well in poly-D-Lysine coated ninety-six well plates(#35-4461; Becton-Dickinson, Bedford, Mass.). Cells were fixed withformalin (Sigma HT50-1-1 Formalin solution, diluted 1:1 with PBS/0.5%BSA before use) by removing cell growth medium, adding 0.2 ml/wellformalin solution and incubating at room temperature for 25 minutes andthen washing one time with 100 ul/well of PBS/0.5% BSA. Wells wereblocked by addition of 0.3 ml/well of BSA blocker (50-61-00; KPL 10% BSADiluent/Blocking Solution, diluted 1:1 with PBS; KPL, Gaithersburg, Md.)followed by incubation at room temperature, with shaking, for 3 hr.Plates were washed 2 times with 1×KP Wash Buffer (50-63-00; KPL).Samples were diluted in Dilution Buffer (PBS/0.5% Tween-20) or DilutionBuffer with 1% Male Lewis Rat Serum (RATSRM-M; Bioreclamation Inc.,Hicksville, N.Y.) and 0.1 ml/well was added to blocked plates,incubating for 1 hr at room temperature with shaking. Plates were washed3 times with 1×KP Wash Buffer and then incubated with HRP-Goatanti-human IgG Fc (#31416; Pierce, Rockford, Ill.) diluted 1:5000 inPBS/0.1% Tween-20 for 1 hr at room temperature, with shaking. Plateswere washed plates 3 30 times with 1×KP Wash Buffer, and then 0.1ml/well TMB substrate (52-00-01; KPL) was added. The reactions werestopped by addition of 0.1 ml/well 2 N Sulfuric Acid. Absorbance wasread at 450 nm on a Molecular Devices SpectroMax 340 (Sunnyvale,Calif.).

FIG. 34B. Whole cell immunoassay was performed as above with thefollowing modifications. HEK 293 cells were plated at 1×10e5 cells/welland HEK Kv1.3 cells were plated at 6×10e4 cells/well in poly-D-Lysinecoated 96 well plates. Fc Control was added at 500 ng/ml in a volume of0.05 ml/well. HRP-Goat anti-human IgG Fc (#31416; Pierce, Rockford,Ill.) was diluted 1:10,000 in PBS/0.1% Tween-20. ABTS (50-66-00, KPL)was used as the substrate. Absorbances were read at 405 nm afterstopping reactions by addition of 0.1 ml/well of 1% SDS.

Example 38 Purification of Fc-L10-ShK(1-35)

Expression of Fc-L10-ShK[1-35] was as described in Example 3 hereinabove. Frozen, E. coli paste (18 g) was combined with 200 ml of roomtemperature 50 mM tris HCl, 5 mM EDTA, pH 8.0 and was brought to about0.1 mg/ml hen egg white lysozyme. The suspended paste was passed througha chilled microfluidizer twice at 12,000 PSI. The cell lysate was thencentrifuged at 22,000 g for 15 min at 4° C. The pellet was thenresuspended in 200 ml 1% deoxycholic acid using a tissue grinder andthen centrifuged at 22,000 g for 15 min at 4° C. The pellet was thenresuspended in 200 ml water using a tissue grinder and then centrifugedat 22,000 g for 15 min at 4° C. The pellet (3.2 g) was then dissolved in32 ml 8 M guanidine HCl, 50 mM tris HCl, pH 8.0. The pellet solution wasthen centrifuged at 27,000 g for 15 min at room temperature, and then 5ml of the supernatant was transferred to 500 ml of the refolding buffer(3 M urea, 20% glycerol, 50 mM tris, 160 mM arginine HCl, 5 mM EDTA, 1mM cystamine HCl, 4 mM cysteine, pH 9.5) at 4° C. with vigorousstirring. The stirring rate was then slowed and the incubation wascontinued for 2 days at 4° C. The refolding solution was then stored at−70° C.

The stored refold was defrosted and then diluted with 2 L of water andthe pH was adjusted to 7.3 using 1 M H₃PO₄. The pH adjusted material wasthen filtered through a 0.22 μm cellulose acetate filter and loaded onto a 60 ml Amersham SP-FF (2.6 cm I.D.) column at 20 ml/min in S-BufferA (20 mM NaH2PO4, pH 7.3) at 7° C. The column was then washed withseveral column volumes of S-Buffer A, followed by elution with a lineargradient from 0% to 60% S-Buffer B (20 mM NaH2PO4, 1 M NaCl, pH 7.3)followed by a step to 100% S-Buffer B at 10 ml/min 7° C. Fractions werethen analyzed using a Coomassie brilliant blue stained tris-glycine4-20% SDS-PAGE, and the fractions containing the desired product werepooled based on these data. The pool was then loaded on to a 1 mlAmersham rProtein A HiTrap column in PBS at 1 ml/min 7° C. Then columnwas then washed with several column volumes of 20 mM NaH₂PO₄ pH 6.5, 1 MNaCl and eluted with 100 mM glycine pH 3.0. To the elution peak, 0.0125volumes (25 ml) of 3 M sodium acetate was added.

A spectral scan was then conducted on 50 μl of the combined pool dilutedin 700 μl water using a Hewlett Packard 8453 spectrophotometer (FIG.46A). The concentration of the filtered material was determined to be2.56 mg/ml using a calculated molecular mass of 30,410 g/mol andextinction coefficient of 36,900 M⁻¹ cm⁻¹. The purity of the filteredmaterial was then assessed using a Coomassie brilliant blue stainedtris-glycine 4-20% SDS-PAGE (FIG. 46B). The macromolecular state of theproduct was then determined using size exclusion chromatography on 20 μgof the product injected on to a Phenomenex BioSep SEC 3000 column(7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observingthe absorbance at 280 nm (FIG. 46C). The product was then subject tomass spectral analysis by diluting 1 μl of the sample into 10 μl ofsinapinic acid (10 mg per ml in 0.05% trifluroacetic acid, 50%acetonitrile). One milliliter of the resultant solution was spotted ontoa MALDI sample plate. The sample was allowed to dry before beinganalyzed using a Voyager DE-RP time-of-flight mass spectrometer equippedwith a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear modewas used, with an accelerating voltage of 25 kV. Each spectrum wasproduced by accumulating data from ˜200 laser shots. External masscalibration was accomplished using purified proteins of known molecularmasses. The product was then stored at −80° C.

The IC₅₀ for blockade of human Kv1.3 by purified E. coli-derivedFc-L10-ShK[1-35], also referred to as “Fc-L-ShK[1-35]”, is shown inTable 35 (in Example 50 herein below).

Example 39 Purification of Bacterially Expressed Fc-L10-ShK(2-35)

Expression of Fc-L10-ShK[2-35] was as described in Example 4 hereinabove. Frozen, E. coli paste (16.5 g) was combined with 200 ml of roomtemperature 50 mM tris HCl, 5 mM EDTA, pH 8.0 and was brought to about0.1 mg/ml hen egg white lysozyme. The suspended paste was passed througha chilled microfluidizer twice at 12,000 PSI. The cell lysate was thencentrifuged at 22,000 g for 15 min at 4° C. The pellet was thenresuspended in 200 ml 1% deoxycholic acid using a tissue grinder andthen centrifuged at 22,000 g for 15 min at 4° C. The pellet was thenresuspended in 200 ml water using a tissue grinder and then centrifugedat 22,000 g for 15 min at 4° C. The pellet (3.9 g) was then dissolved in39 ml 8 M guanidine HCl, 50 mM tris HCl, pH 8.0. The pellet solution wasthen centrifuged at 27,000 g for 15 min at room temperature, and then 5ml of the supernatant was transferred to 500 ml of the refolding buffer(3 M urea, 20% glycerol, 50 mM tris, 160 mM arginine HCl, 5 mM EDTA, 1mM cystamine HCl, 4 mM cysteine, pH 9.5) at 4° C. with vigorousstirring. The stirring rate was then slowed and the incubation wascontinued for 2 days at 4° C. The refolding solution was then stored at−70° C.

The stored refold was defrosted and then diluted with 2 L of water andthe pH was adjusted to 7.3 using 1 M H₃PO₄. The pH adjusted material wasthen filtered through a 0.22 μm cellulose acetate filter and loaded onto a 60 ml Amersham SP-FF (2.6 cm I.D.) column at 20 ml/min in S-BufferA (20 mM NaH₂PO₄, pH 7.3) at 7° C. The column was then washed withseveral column volumes of S-Buffer A, followed by elution with a lineargradient from 0% to 60% S-Buffer B (20 mM NaH2PO4, 1 M NaCl, pH 7.3)followed by a step to 100% S-Buffer B at 10 ml/min 7° C. The fractionscontaining the desired product were pooled and filtered through a 0.22μm cellulose acetate filter. The pool was then loaded on to a 1 mlAmersham rProtein A HiTrap column in PBS at 2 ml/min 7° C. Then columnwas then washed with several column volumes of 20 mM NaH₂PO₄ pH 6.5, 1 MNaCl and eluted with 100 mM glycine pH 3.0. To the elution peak, 0.0125volumes (18 ml) of 3 M sodium acetate was added, and the sample wasfiltered through a 0.22 μm cellulose acetate filter.

A spectral scan was then conducted on 20 μl of the combined pool dilutedin 700 μl water using a Hewlett Packard 8453 spectrophotometer (FIG.40A). The concentration of the filtered material was determined to be3.20 mg/ml using a calculated molecular mass of 29,282 g/mol andextinction coefficient of 36,900 M⁻¹ cm⁻¹. The purity of the filteredmaterial was then assessed using a Coomassie brilliant blue stainedtris-glycine 4-20% SDS-PAGE (FIG. 40B). The macromolecular state of theproduct was then determined using size exclusion chromatography on 50 μgof the product injected on to a Phenomenex BioSep SEC 3000 column(7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observingthe absorbance at 280 nm (FIG. 40C). The product was then subject tomass spectral analysis by diluting 1 μl of the sample into 10 μl ofsinapinic acid (10 mg per ml in 0.05% trifluroacetic acid, 50%acetonitrile). One milliliter of the resultant solution was spotted ontoa MALDI sample plate. The sample was allowed to dry before beinganalyzed using a Voyager DE-RP time-of-flight mass spectrometer equippedwith a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear modewas used, with an accelerating voltage of 25 kV. Each spectrum wasproduced by accumulating data from ˜200 laser shots. External masscalibration was accomplished using purified proteins of known molecularmasses (FIG. 40D). The product was then stored at −80° C.

The IC₅₀ for blockade of human Kv1.3 by purified E. coli-derivedFc-L10-ShK[2-35], also referred to as “Fc-L-ShK[2-35]”, is shown inTable 35 (in Example 50 herein below).

Example 40 Purification of Bacterially Expressed Fc-L10-OsK1

Frozen, E. coli paste (129 g; see Example 10) was combined with 1290 mlof room temperature 50 mM tris HCl, 5 mM EDTA, pH 7.8 and was brought toabout 0.1 mg/ml hen egg white lysozyme. The suspended paste was passedthrough a chilled microfluidizer twice at 12,000 psi. The cell lysatewas then centrifuged at 17,700 g for 15 min at 4° C. The pellet was thenresuspended in 1290 ml 1% deoxycholic acid using a tissue grinder andthen centrifuged at 17,700 g for 15 min at 4° C. The pellet was thenresuspended in 1290 ml water using a tissue grinder and then centrifugedat 17,700 g for 15 min at 4° C. 8 g of the pellet (16.3 g total) wasthen dissolved in 160 ml 8 M guanidine HCl, 50 mM tris HCl, pH 8.0. 100ml of the pellet solution was then incubated with 1 ml of 1 M DTT for 60min at 37° C. The reduced material was transferred to 5000 ml of therefolding buffer (1 M urea, 50 mM tris, 160 mM arginine HCl, 2.5 mMEDTA, 1.2 mM cystamine HCl, 4 mM cysteine, pH 10.5) at 2 ml/min, 4° C.with vigorous stirring. The stirring rate was then slowed and theincubation was continued for 3 days at 4° C.

The pH of the refold was adjusted to 8.0 using acetic acid. The pHadjusted material was then filtered through a 0.22 μm cellulose acetatefilter and loaded on to a 50 ml Amersham Q Sepharose-FF (2.6 cm I.D.)column at 10 ml/min in Q-Buffer A (20 mM Tris, pH 8.5) at 8° C. with aninline 50 Amersham Protein A column (2.6 cm I.D.). After loading, the QSepharose column was removed from the circuit, and the remainingchromatography was carried out on the protein A column. The column waswashed with several column volumes of Q-Buffer A, followed by elutionusing a step to 100 mM glycine pH 3.0. The fractions containing thedesired product were pooled and immediately loaded on to a 50 mlAmersham SP-Sepharose HP column (2.6 cm I.D.) at 20 ml/min in S-Buffer A(20 mM NaH₂PO₄, pH 7.0) at 8° C. The column was then washed with severalcolumn volumes of S-Buffer A followed by a linear gradient from 5% to60% S-Buffer B (20 mM NaH₂PO₄, 1 M NaCl, pH 7.0) followed by a step to100% S-Buffer B. Fractions were then analyzed using a Coomassiebrilliant blue stained tris-glycine 4-20% SDS-PAGE. The fractionscontaining the bulk of the desired product were pooled and then appliedto a 75 ml MEP Hypercel column (2.6 cm I.D.) at 5 ml/min in MEP Buffer A(20 mM tris, 200 mM NaCl, pH 8.0) at 8° C. Column was eluted with alinear gradient from 5% to 50% MEP Buffer B (50 mM sodium citrate pH4.0) followed by a step to 100% MEP Buffer B. Fractions were thenanalyzed using a Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE, and the fractions containing the bulk of the desired productwere pooled.

The MEP pool was then concentrated to about 20 ml using a Pall Jumbo-Sepwith a 10 kDa membrane followed by buffer exchange with FormulationBuffer (20 mM NaH₂PO₄, 200 mM NaCl, pH 7.0) using the same membrane. Aspectral scan was then conducted on 50 μl of the combined pool dilutedin 700 μl Formulation Buffer using a Hewlett Packard 8453spectrophotometer (FIG. 41A). The concentration of the material wasdetermined to be 4.12 mg/ml using a calculated molecular mass of 30,558g/mol and extinction coefficient of 35,720 M⁻¹ cm⁻¹. The purity of thematerial was then assessed using a Coomassie brilliant blue stainedtris-glycine 4-20% SDS-PAGE (FIG. 41B). The macromolecular state of theproduct was then determined using size exclusion chromatography on 123μg of the product injected on to a Phenomenex BioSep SEC 3000 column(7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observingthe absorbance at 280 nm (FIG. 41C). The product was then subject tomass spectral analysis by chromatographing approximately 4 μg of thesample through a RP-HPLC column (Vydac C₄, 1×150 mm). Solvent A was 0.1%trifluoroacetic acid in water and solvent B was 0.1% trifluoroaceticacid in 90% acetonitrile, 10% water. The column was pre-equilibrated in10% solvent B at a flow rate of 80 μl per min. The protein was elutedusing a linear gradient of 10% to 90% solvent B over 30 min. Part of theeffluent was directed into a LCQ ion trap mass spectrometer. The massspectrum was deconvoluted using the Bioworks software provided by themass spectrometer manufacturer. (FIG. 41D). The product was filteredthrough a 0.22 μm cellulose acetate filter and then stored at −80° C.

The yield for the E. coli-expressed Fc-L10-OSK1 prep was 81 mg from 40 gof cell paste (129 g×(8 g/16.3 g)×(100 ml/160 ml)=39.6 g which wasrounded to 40 g), the purity was greater than 80% judging by SDS-PAGE,it is running as the expected dimer judging by SEC-HPLC, and the masswas within the expected molecular weight range judging by MS.

The IC₅₀ for blockade of human Kv1.3 by purified E. coli-derivedFc-L10-OSK1, also referred to as “Fc-L-OSK1”, is shown in Table 35 (inExample 50 herein below).

Example 41 Fc-L10-OSK1, Fc-L10-OSK1 [K7S], Fc-L10-OSK1 [E16K,K20D], andFc-L10-OSK1 [K7S,E16K,K20D] Expressed by Mammalian Cells

Fc-L10-OSK1, Fc-L10-OSK1 [K7S], Fc-L10-OSK1 [E16K,K20D], and Fc-L10-OSK1[K7S,E16K,K20D], inhibitors of Kv1.3, were expressed in mammalian cells.A DNA sequence coding for the Fc region of human IgG1 fused in-frame toa linker sequence and a monomer of the Kv1.3 inhibitor peptide OSK1,OSK1 [K7S], OSK1 [E16K,K20D], or OSK1 [K7S,E16K,K20D] was constructed asdescribed below. Methods for expressing and purifying the peptibody frommammalian cells (HEK 293 and Chinese Hamster Ovary cells) are disclosedherein.

For construction of Fc-L10-OSK1, Fc-L10-OSK1 [K7S], Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1 [K7S,E16K,K20D] expression vectors, a PCRstrategy was employed to generate the full length genes, OSK1, OSK1[K7S], OSK1 [E16K,K20D], and OSK1 [K7S,E16K,K20D], each linked to a fourglycine and one serine amino acid linker with two stop codons andflanked by BamHI and NotI restriction sites as shown below.

Two oligos for each of OSK1, OSK1 [K7S], OSK1 [E16K,K20D], and[K7S,E16K,K20D]OSK1 with the sequence as depicted below were used in aPCR reaction with Pfu Turbo HotStart DNA polymerase (Stratagene) at 95°C.-30 sec, 55° C.-30 sec, 75° C.-45 sec for 35 cycles; BamHI (ggatcc)and NotI (gcggccgc) restriction sites are underlined.

OSK1: Forward primer: cat gga tcc gga gga gga gga agc (SEQ ID NO: 876)ggc gtg atc atc aac gtg aag tgc aag atc agc cgc cag tgc ctg gagccc tgc aag aag gcc g; Reverse primer: cat gcg gcc gct tac tac ttg ggg(SEQ ID NO: 877) gtg cag tgg cac ttg ccg ttc atgcac ttg ccg aag cgc atg ccg gcc ttc ttg cag ggc tcc a; OSK1[K7S]:Forward primer: cat gga tcc gga gga gga gga agc (SEQ ID NO: 878)ggc gtg atc atc aac gtg agc tgc aag atc agc cgc cag tgc ctg gagccc tgc aag aag gcc g; Reverse primer: cat gcg gcc gct tac tac ttg ggg(SEQ ID NO: 879) gtg cag tgg cac ttg ccg ttc atgcac ttg ccg aag cgc atg ccg gcc ttc ttg cag ggc tcc a; OSK1[E16K, K20D]:Forward primer: cat gga tcc gga gga gga gga agc (SEQ ID NO: 880)ggc gtg atc atc aac gtg aag tgc aag atc agc cgc cag tgc ctg aagccc tgc aag gac gcc g; Reverse primer: cat gcg gcc gct tac tac ttg ggg(SEQ ID NO: 881) gtg cag tgg cac ttg ccg ttc atgcac ttg ccg aag cgc atg ccg gcg tcc ttg cag ggc ttc a;OSK1[K7S, E16K, K20D]: Forward primer: cat gga tcc gga gga gga gga agc(SEQ ID NO: 882) ggc gtg atc atc aac gtg agc tgcaag atc agc cgc cag tgc ctg aag ccc tgc aag gac gcc g; Reverse primer:cat gcg gcc gct tac tac ttg ggg (SEQ ID NO: 883)gtg cag tgg cac ttg ccg ttc atg cac ttg ccg aag cgc atg ccg gcgtcc ttg cag ggc ttc a.

The resulting PCR products were resolved as the 155 bp bands on a fourpercent agarose gel. The 155 bp PCR product was purified using PCRPurification Kit (Qiagen), then digested with BamHI and NotI (Roche)restriction enzymes, and agarose gel was purified by Gel Extraction Kit(Qiagen). At the same time, the pcDNA3.1 (+)CMVi-hFc-Shk[2-35] vectorwas digested with BamHI and NotI restriction enzymes and the largefragment was purified by Gel Extraction Kit. The gel purified PCRfragment was ligated to the purified large fragment and transformed intoOne Shot® Top 10F′ (Invitrogen). DNAs from transformed bacterialcolonies were isolated and digested with BamHI and NotI restrictionenzymes and resolved on a two percent agarose gel. DNAs resulting in anexpected pattern were submitted for sequencing. Although, analysis ofseveral sequences of clones yielded a 100% percent match with the abovesequences, only one clone from each gene was selected for large scaledplasmid purification. The DNA of Fc-L10-OSK1, Fc-L10-OSK1[K7S],Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1[K7S,E16K,K20D] in pCMVi vectorwas resequenced to confirm the Fc and linker regions and the sequencewas 100% identical to the above sequences. The sequences and pictorialrepresentations of Fc-L10-OSK1, Fc-L10-OSK1[K7S],Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1[K7S,E16K,K20D] are depicted inFIG. 42A-B, FIG. 43A-B, FIG. 44A-B and FIG. 45A-B, respectively.

HEK-293 cells used in transient transfection expression of Fc-L10-OSK1,Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1[K7S,E16K,K20D]in pCMVi protein were cultured in growth medium containing DMEM HighGlucose (Gibco), 10% fetal bovine serum (FBS from Gibco), 1×non-essential amino acid (NEAA from Gibco) and 1×Penicillin/Streptomycine/Glutamine (Pen/Strep/Glu from Gibco). 5.6 μgeach of Fc-L10-OSK1, Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K,K20D], andFc-L10-OSK1[K7S,E16K,K20D] in pCMVi plasmid that had beenphenol/chloroform extracted was transfected into HEK-293 cells usingFuGENE 6 (Roche). The cells were recovered for 24 hours, and then placedin DMEM High Glucose, 1×NEAA and 1× Pen/Strep/Glu medium for 48 hours.Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1[K7S,E16K,K20D]were purified from medium conditioned by these transfected HEK-293 cellsusing a protocol described in Example 50 herein below.

Fifteen μl of conditioned medium was mixed with an in-house 4× LoadingBuffer (without β-mercaptoethanol) and electrophoresed on a Novex 4-20%tris-glycine gel using a Novex Xcell II apparatus at 101V/46 mA for 2hours in a 1× Gel Running solution (25 mM Tris Base, 192 mM Glycine, 3.5mM SDS) along with 20 μl of BenchMark Pre-Stained Protein ladder(Invitrogen). The gel was then soaked in Electroblot buffer (25 mM Trisbase, 192 mM glycine, 20% methanol,) for 5 minutes. A nitrocellulosemembrane from Invitrogen (Cat. No. LC200, 0.2 μm pores size) was soakedin Electroblot buffer. The pre-soaked gel was blotted to thenitrocellulose membrane using the Mini Trans-Blot Cell module accordingto the manufacturer instructions (Bio-Rad Laboratories) at 300 mA for 2hours. The blot was rinsed in Tris buffered saline solution pH 7.5 with0.1% Tween20 (TBST). Then, the blot was first soaked in a 5% milk(Carnation) in TBST for 1 hour at room temperature, followed by washingthree times in TBST for 10 minutes per wash. Then, incubated with 1:1000dilution of the HRP-conjugated Goat anti-human IgG, (Fcγ) antibody(Pierece Biotechnology Cat. no. 31413) in TBST with 5% milk buffer for 1hour with shaking at room temperature. The blot was then washed threetimes in TBST for 15 minutes per wash at room temperature. The primaryantibody was detected using Amersham Pharmacia Biotech's ECL westernblotting detection reagents according to manufacturer's instructions.Upon ECL detection, the western blot analysis displayed the expectedsize of 66 kDa under non-reducing gel conditions (FIG. 46).

Plasmids containing the Fc-L10-OSK1, Fc-L10-OSK1[K7S],Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1[K7S,E16K,K20D] inserts in pCMVivector were digested with XbaI and NotI (Roche) restriction enzymes andgel purified. The inserts were individually ligated into SpeI and NotI(Roche) digested pDSRα24 (Amgen Proprietary) expression vector.Integrity of the resulting constructs were confirmed by DNA sequencing.Although, analysis of several sequences of clones yielded a 100% percentmatch with the above sequence, only one clone was selected for largescaled plasmid purification.

AM1 CHOd-(Amgen Proprietary) cells used in the stable expression ofFc-L10-OSK1 protein were cultured in AM1 CHOd-growth medium containingDMEM High Glucose, 10% fetal bovine serum, lx hypoxantine/thymidine (HTfrom Gibco), 1×NEAA and 1× Pen/Strep/Glu. 5.6 μg of pDSRα-24-Fc-L10-OSK1plasmid was transfected into AM1 CHOd-cells using FuGene 6. Twenty-fourhours post transfection, the cells were split 1:11 into DHFR selectionmedium (DMEM High Glucose plus 10% Dialyzed Fetal Bovine Serum (dFBS),1×NEAA and 1× Pen/Strep/Glu) and at 1:50 dilution for colony selection.The cells were selected in DHFR selection medium for thirteen days. Theten 10-cm² pools of the resulting colonies were expanded to ten T-175flasks, then were scaled up ten roller bottles and cultured under AM1CHOd-production medium (DMEM/F12 (1:1), 1×NEAA, 1× Sodium Pyruvate (NaPyruvate), 1× Pen/Strep/Glu and 1.5% DMSO). The conditioned medium washarvested and replaced at one-week intervals. The resulting six litersof conditioned medium were filtered through a 0.45 μm cellulose acetatefilter (Corning, Acton, Mass.), and characterized by SDS-PAGE analysisas shown in FIG. 47. Then, transferred to Protein Chemistry forpurification.

Twelve colonies were selected after 13 days on DHFR selection medium andpicked into one 24-well plate. The plate was allowed to grow up for oneweek, and then was transferred to AM1 CHOd-production medium for 48-72hours and the conditioned medium was harvested. The expression levelswere evaluated by Western blotting similar to the transient Western blotanalysis with detection by the same HRP-conjugated Goat anti-human IgG,(Fcγ) antibody to screen 5 μl of conditioned medium. All 12 stableclones exhibited expression at the expected size of 66 kDa. Two clones,A3 and C2 were selected and expanded to T175 flask for freezing with A3as a backup to the primary clone C2 (FIG. 48).

The C2 clone was scaled up into fifty roller bottles (Corning) usingselection medium and grown to confluency. Then, the medium was exchangedwith a production medium, and let incubate for one week. The conditionedmedium was harvested and replaced at the one-week interval. Theresulting fifty liters of conditioned medium were filtered through a0.45 μm cellulose acetate filter (Corning, Acton, Mass.), andcharacterized by SDS-PAGE analysis (data not shown). Furtherpurification was accomplished as described in Example 42 herein below.

Example 42 Purification of Fc-L10-OSK1, Fc-10-OSK1(K7S),Fc-L10-OSK1(E16K,K20D), and Fc-L10-OSK1(K7S,E16K,K20D) Expressed byMammalian Cells

Purification of Fc-L10-OSK1. Approximately 6 L of CHO (AM1 CHOd-)cell-conditioned medium (see, Example 41 above) was loaded on to a 35 mlMAb Select column (GE Healthcare) at 10 ml/min 7° C., and the column waswashed with several column volumes of Dulbecco's phosphate bufferedsaline without divalent cations (PBS) and sample was eluted with a stepto 100 mM glycine pH 3.0. The MAb Select elution was directly loaded onto an inline 65 ml SP-HP column (GE Healthcare) in S-Buffer A (20 mMNaH₂PO₄, pH 7.0) at 10 ml/min 7° C. After disconnecting the MAb selectcolumn, the SP-HP column was then washed with several column volumesS-Buffer A, and then developed using a linear gradient from 5% to 60%S-Buffer B (20 mM NaH₂PO₄, 1 M NaCl, pH 7.0) at 10 ml/min followed by astep to 100% S-Buffer B at 7° C. Fractions were then analyzed using aCoomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE, and thefractions containing the desired product were pooled based on thesedata. The pooled material was then concentrated to about 20 ml using aPall Life Sciences Jumbosep 10K Omega centrifugal ultra-filtrationdevice. The concentrated material was then buffer exchanged by dilutingwith 20 ml of 20 mM NaH₂PO₄, pH 7.0 and reconcentrated to 20 ml usingthe Jumbosep 10K Omega filter. The material was then diluted with 20 ml20 mM NaH₂PO₄, 200 mM NaCl, pH 7.0 and then reconcentrated to 22 ml. Thebuffer exchanged material was then filtered though a Pall Life SciencesAcrodisc with a 0.22 μm, 25 mm Mustang E membrane at 1 ml/min roomtemperature. A spectral scan was then conducted on 50 μl of the filteredmaterial diluted in 700 μl PBS using a Hewlett Packard 8453spectrophotometer (FIG. 49A, black trace). The concentration of thefiltered material was determined to be 4.96 mg/ml using a calculatedmolecular mass of 30,371 g/mol and extinction coefficient of 35,410 M⁻¹cm⁻¹. The purity of the filtered material was then assessed using aCoomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 49B).The endotoxin level was then determined using a Charles RiverLaboratories Endosafe-PTS system (0.05-5 EU/ml sensitivity) using a30-fold dilution of the sample in Charles Rivers Laboratories EndotoxinSpecific Buffer yielding a result of 1.8 EU/mg protein. Themacromolecular state of the product was then determined using sizeexclusion chromatography on 149 μg of the product injected on to aPhenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mMNaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 49C).The product was then subject to mass spectral analysis by diluting 1 μlof the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05%trifluroacetic acid, 50% acetonitrile). One milliliter of the resultantsolution was spotted onto a MALDI sample plate. The sample was allowedto dry before being analyzed using a Voyager DE-RP time-of-flight massspectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). Thepositive ion/linear mode was used, with an accelerating voltage of 25kV. Each spectrum was produced by accumulating data from about 200 lasershots. External mass calibration was accomplished using purifiedproteins of known molecular masses. (FIG. 49D). The product was thenstored at −80° C.

The yield for the mammalian Fc-L10-OSK1 prep was 115 mg from 6 L, thepurity was >90% judging by SDS-PAGE; Fc-L10-OSK1 ran as the expecteddimer judging by SEC-HPLC, and the mass is with the expected rangejudging by MS.

The activity of purified Fc-L10-OSK1 in blocking human Kv1.3 and humanKv1.1 is described in Example 43 herein below.

Purification of Fc-L10-OSK1(K7S), Fc-L10-OSK1(E16K,K20D), andFc-L10-OSK1(K7S,E16K,K20D). Approximately 500 mL of medium conditionedby transfected HEK-293 (see, Example 41 above) was combined with a 65%slurry of MAb Select resin (1.5 ml) (GE Healthcare) and 500 μl 20% NaN₃.The slurry was then gently agitated for 3 days at 4° C. followed bycentrifugation at 1000 g for 5 minutes at 4° C. using no brake. Themajority of the supernatant was then aspirated and the remaining slurryin the pellet was transferred to a 14 ml conical tube and combined with12 ml of Dulbecco's phosphate buffered saline without divalent cations(PBS). The slurry was centrifuged at 2000 g for 1 minute at 4° C. usinga low brake and the supernatant was aspirated. The PBS wash cycle wasrepeated an additional 3 times. The bound protein was then eluted byadding 1 ml of 100 mM glycine pH 3.0 and gently agitating for 5 min atroom temperature. The slurry was then centrifuged at 2000 g for 1 minuteat 4° C. using a low brake and the supernatant was aspirated as thefirst elution. The elution cycle was repeated 2 more times, and all 3supernatants were combined into a single pool. Sodium acetate (37.5 μlof a 3 M solution) was added to the elution pool to raise the pH, whichwas then dialyzed against 10 mM acetic acid, 5% sorbitol, pH 5.0 for 2hours at room temperature using a 10 kDa SlideAlyzer (Pierce). Thedialysis buffer was changed, and the dialysis continued over night at 4°C. The dialyzed material was then filtered through a 0.22 μm celluloseacetate filter syringe filter. Then concentration of the filteredmaterial was determined to be 1.27 mg/ml using a calculated molecularmass of 30,330 and extinction coefficient of 35,410 M⁻¹ cm⁻¹ (FIG. 50A).The purity of the filtered material was then assessed using a Coomassiebrilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 50B). Theendotoxin level was then determined using a Charles River LaboratoriesEndosafe-PTS system (0.05-5 EU/ml sensitivity) using a 25-fold dilutionof the sample in Charles Rivers Laboratories Endotoxin Specific Bufferyielding a result of <1 EU/mg protein. The macromolecular state of theproduct was then determined using size exclusion chromatography on 50 μgof the product injected on to a Phenomenex BioSep SEC 3000 column(7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observingthe absorbance at 280 nm (FIG. 50C). The product was then subject tomass spectral analysis by diluting 1 μl of the sample into 10 μl ofsinapinic acid (10 mg per ml in 0.05% trifluroacetic acid, 50%acetonitrile). One milliter of the resultant solution was spotted onto aMALDI sample plate. The sample was allowed to dry before being analyzedusing a Voyager DE-RP time-of-flight mass spectrometer equipped with anitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode wasused, with an accelerating voltage of 25 kV. Each spectrum was producedby accumulating data from ˜200 laser shots. External mass calibrationwas accomplished using purified proteins of known molecular masses.(FIG. 50D). The product was then stored at −80° C.

FIGS. 51A-D show results from the purification and analysis forFc-L10-OsK1(E16K, K20D), which was conducted using the same protocol asthat for the Fc-L10-OsK1(K7S) molecule (described above) with thefollowing exceptions: the concentration was found to be 1.59 mg/ml usinga calculated molecular mass of 30,357 g/mol and a calculated extinctioncoefficient of 35,410; the pyrogen level was found to be <1 EU/mg usinga 32-fold dilution.

FIGS. 52A-D show results from the purification and analysis forFc-L10-OsK1(K7S,E16K, K20D), which was conducted using the same protocolas that for the Fc-L10-OsK1(K7S) molecule (described above) with thefollowing exceptions: the concentration was found to be 0.81 mg/ml usinga calculated molecular mass of 30,316 g/mol and a calculated extinctioncoefficient of 35,410; the pyrogen level was found to be <1 EU/mg usinga 16-fold dilution.

The activity of purified Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K, K20D] andFc-L10-OSK1[K7S, E16K, K20D] in blocking human Kv1.3 and human Kv1.1 isdescribed in Example 43 herein below.

Example 43 Electrophysiology of OSK1 and OSK1 Peptibody Analogs

A 38-residue peptide toxin of the Asian scorpion Orthochirusscrobiculosus venom (OSK1) was synthesized (see, Examples 41) toevaluate its impact on the human Kv1.1 and Kv1.3 channels, subtypes ofthe potassium channel family. The potency and selectivity of syntheticOSK1 in inhibiting the human Kv1.1 and Kv1.3 channels was evaluated bythe use of HEK293 cell expression system and electrophysiology (FIG.53). Whole cell patch clamp recording of stably expressed Kv1.3 channelsrevealed that the synthetic OSK1 peptide is more potent in inhibitinghuman Kv1.3 when compared to Kv1.1 (Table 33).

Fusion of OSK1 peptide toxin to antibody to generate OSK1 peptibody. Toimprove plasma half-life and prevent OSK1 peptide toxin from penetratingthe CNS, the OSK1 peptide toxin was fused to the Fc-fragment of a humanantibody IgG1 via a linker chain length of 10 amino acid residues(Fc-L10-OSK1), as described in Example 41 herein. This fusion resultedin a decrease in the potency of Kv1.3 by 5-fold when compared to thesynthetic OSK1 peptide. However, it significantly improved theselectivity of OSK1 against Kv1.1 by 210-fold when compared to that ofthe synthetic peptide alone (4-fold; Table 33 and FIG. 54).

Modification of OSK1-peptibody (Fc-L10-OSK1). OSK1 shares 60 to 80%sequence homology to other members of scorpion toxins, which arecollectively termed α-KTx3. Sequence alignment of OSK1 and other membersof α-KTx3 family revealed 4 distinct structural differences at positions12, 16, 20, and 36. These structural differences of OSK1 have beenpostulated to play an important role in its wide range of activitiesagainst other potassium channels, which is not observed with othermembers of α-KTx3 family. Hence, two amino acid residues at position 16and 20 were restored to the more conserved amino acid residues withinthe OSK1 sequence in order to evaluate their impact on selectivityagainst other potassium channels such as Kv1.1, which is predominantlyfound in the CNS as a heterotetromer with Kv1.2. By substituting forglutamic acid at position 16, and for lysine at position 20, theconserved lysine and aspartic acid residues, respectively (i.e.,Fc-L10-OSK1[E16K, K20D]), we did not observe a significant change inpotency when compared to that of Fc-L10-OSK1 (1.3-fold difference; FIG.56 and Table 33). However, this double mutation removed the blockingactivity against Kv1.1. The selectivity ratio of Kv1.1/Kv1.3 was403-fold, which was a significant improvement over the selectivity ratiofor Fc-L10-OSK1 (210-fold). A single amino acid mutation at position 7from lysine to serine (Fc-L10-OSK1[K7S]) produced a slight change inpotency and selectivity by 2- and 1.3-fold, respectively, when comparedto those of Fc-L10-OSK1 (FIG. 55 and Table 33). There was a significantdecrease in potency as well as selectivity when all three residues weremutated to generate Fc-L10-OSK1[K7S, E16K, K20D] (FIG. 57 and Table 33).

As demonstrated by the results in Table 33, we dramatically improvedselectivity against Kv1.1 by fusing the OSK1 peptide toxin to theFc-fragment of the human antibody IgG1, but reduced target potencyagainst Kv1.3. The selectivity against Kv1.1 was further improved when 2residues at two key positions were restored to the conserved residuesfound in other members of the α-KTx3 family.

TABLE 33 shows a summary of IC50 values for OSK1 and OSK1 analoguesagainst hKv1.3 and hKv1.1 channels. All analogues are ranked based ontheir potency against hKv1.3. Also shown in the table is the selectivityratio of hKv1.1/hKv1.3 for all OSK1 analogues. hKv1.3:IC₅₀ hKv1.1:IC₅₀hKv1.1/ Compound [pM] [pM] hKv1.3 Synthetic OSK1 39 160 4 FC-L10-OSK1198 41600 210 Fc-L10-OSK1[E16K, 248 100000 403 K20D] Fc-L10-OSK1[K7S]372 100000 269 Fc-L10-OSK1[K7S, 812 10000 12 E16K, K20D]

Example 44 Pharmacokinetic Study of PEG-ShK[1-35] Molecule in Rats

The intravenous (IV) pharmacokinetic profile was determined of a about24-kDa 20K PEG-ShK[1-35] molecule and the about 4-kDa small native ShKpeptide was determined in Spraque Dawley rats. The IV dose for thenative ShK peptide and our novel 20K PEG-ShK[1-35] molecule was 1 mg/kg.This dose represented equal molar amounts of these two molecules. Theaverage weight of the rats was about 0.3 kg and two rats were used foreach dose & molecule. At various times following IV injection, blood wasdrawn and about 0.1 ml of serum was collected. Serum samples were storedfrozen at −80° C. until analysis.

Assay Plate preparation for electrophysiology. Rat serum samplescontaining the 20K PEG-ShK[1-35] molecule or the native ShK peptide frompharmacokinetic studies were received frozen. Before experiments, eachsample was thawed at room temperature and an aliquot (70 to 80 μl) wastransferred to a well in a 96-well polypropylene plate. In order toprepare the Assay Plate, several dilutions were made from thepharmacokinetic serum samples to give rise to Test Solutions. Dilutionsof serum samples from the pharmacokinetic study were into 10% PhosphateBuffered Saline (PBS, with Ca²⁺ and Mg²⁺). For determination of theamount of our novel 20K PEG-ShK[1-35] molecule in serum samples from thepharmacokinetic study, the final serum concentrations in the TestSolutions were 90%, 30%, 10%, 3.3% and 1.1%. Purified 20K PEG-Shk[1-35]Standard inhibition curves were also prepared in the Assay Plate. To dothis, 8-point serial dilutions of the purified 20K PEG-ShK[1-35]molecule (Standard) were prepared in either 90%, 30%, 10%, 3.3% or 1.1%rat serum and the final concentration of standard was 50, 16.7, 5.5,1.85, 0.62, 0.21, 0.068 and 0.023 nM.

Cell preparation for electrophysiology. CHO cells stably expressing thevoltage-activated K⁺ channel, K_(v)1.3 were plated in T-175 tissueculture flasks (at a density of 5×10⁶) 2 days before experimentation andallowed to grow to around 95% confluence. Immediately prior to theexperiment, the cells were washed with PBS and then detached with a 2 mlmixture (1:1 volume ratio) of trypsin (0.25%) and versene (1:5000) at37° C. (for 3 minutes). Subsequently, the cells were re-suspended in theflask in 10 ml of tissue culture medium (HAM's F-12 with Glutamax,InVitrogen, Cat#31765) with 10% FBS, 1×NEAA and 750 μg/ml of G418) andcentrifuged at about 1000 rpm for 1½ minutes. The resultant cell pelletwas re-suspended in PBS at 3-5×10⁶ cells/ml. IonWorks electrophysiologyand data analysis. The ability of Test solutions or Standards in serumto inhibit K⁺ currents in the CHO-K_(v)1.3 cells was investigated usingthe automated electrophysiology system, IonWorks Quattro. Re-suspendedcells, the Assay Plate, a Population Patch Clamp (PPC) PatchPlate aswell as appropriate intracellular (90 mMK-Gluconate, 20 mMKF, 2 mM NaCl,1 mM MgCl2, 10 mM EGTA, 10 mM HEPES, pH 7.35) and extracellular (PBS,with Ca²⁺ and Mg²⁺) buffers were positioned on IonWorks Quattro.Electrophysiology recordings were made from the CHO-K_(v)1.3 cells usingan amphotericin-based perforated patch-clamp method. Using thevoltage-clamp circuitry of the IonWorks Quattro, cells were held at amembrane potential of −80 mV and voltage-activated K⁺ currents wereevoked by stepping the membrane potential to +30 mV for 400 ms. K⁺currents were evoked under control conditions i.e., in the absence ofinhibitor at the beginning of the experiment and after 10-minuteincubation in the presence of the Test Solution or Standard. The mean K⁺current amplitude was measured between 430 and 440 ms and the data wereexported to a Microsoft Excel spreadsheet. The amplitude of the K⁺current in the presence of each concentration of the Test Solution orStandard was expressed as a percentage of the K⁺ current in controlconditions in the same well.

Standard inhibition curves were generated for each standard in variouslevels of rat serum and expressed as current percent of control (POC)versus log of nM concentration. Percent of control (POC) is inverselyrelated to inhibition, where 100 POC is no inhibition and 0 POC is 100%inhibition. Linear regression over a selected region of the curve wasused to derive an equation to enable calculation of drug concentrationswithin Test solutions. Only current values within the linear portion ofthe Standard curve were used to calculate the concentration of drug inTest solutions. The corresponding Standard curve in a given level ofserum, was always compared to the same level of serum of Test solutionwhen calculating drug level. The Standard curves for ShK and 20KPEG-ShK[1-35] are shown in FIG. 58A and FIG. 58B, respectively, and eachfigure contains linear regression equations for each Standard at a givenpercentage of serum. For the 20K PEG-ShK[1-35] standard curve the linearportion of the Standard curve was from 20 POC to 70 POC and only currentvalues derived from the Test solution which fell within this range wereused to calculate drug concentration within the Test solution.

The pharmacokinetic profile of our novel 20K PEG ShK[1-35] moleculeafter IV injection is shown in FIG. 59. The terminal half-life (t_(1/2)b) of this molecule is estimated from this curve to be between 6 to 12hours long. Beyond 48 hours, the level of drug falls outside the linearrange of the Standard curve and is not calculated. The calculated 6 to12 hour half-life of our novel 20K PEG-ShK[1-35] molecule wassubstantially longer than the approximately 0.33 hour (or 20 min)half-life of the native ShK molecule reported earlier by C. Beeton etal. [C. Beeton et al. (2001) Proc. Natl Acad. Sci. 98, 13942-13947], andis a desirable feature of a therapeutic molecule. A comparison of therelative levels of Kv1.3 inhibitor after an equal molar IV injection ofShK versus 20K PEG-ShK[1-35] is shown in FIG. 60. As can be seen fromthis figure examining 5% serum Test solutions, the 20K PEG-ShK[1-35]molecule showed significant suppression of Kv1.3 current (<70 POC) formore than 24 hours, whereas the native ShK peptide only showed asignificant level of inhibition of Kv1.3 current for the first hour andbeyond 1 hour showed no significant blockade. These data againdemonstrate a desirable feature of the 20K PEG ShK[1-35] molecule as atherapeutic for treatment of autoimmune disease.

Example 45 PEGylated Toxin Peptide Suppressed Severe AutoimmuneEncephalomyelitis in Animal Model

The 20KPEG-ShK inhibitor of Kv1.3 shows improved efficacy in suppressingsevere autoimmune encephalomyelitis in rats. Using an adoptive transferexperimental autoimmune encephalomyelitis (AT-EAE) model of multiplesclerosis described earlier [C. Beeton et al. (2001) J. Immunol. 166,936], we examined the activity in vivo of our novel 20KPEG-ShK moleculeand compared its efficacy to that of the ShK toxin peptide alone. Thestudy design is illustrated in FIG. 61. The results from this in vivostudy are provided in FIG. 62 and FIG. 63. The 20KPEG-ShK moleculedelivered subcutaneously (SC) at 10 μg/kg daily from day −1 to day 3significantly reduced disease severity and increased survival, whereasanimals treated with an equal molar dose (10 μg/kg) of the small ShKpeptide developed severe disease and died.

The 35-amino acid toxin peptide ShK (Stichodactyla helianthusneurotoxin) was purchased from Bachem Bioscience Inc and confirmed byelectrophysiology to potently block Kv1.3 (see Example 36 herein). Thesynthesis, PEGylation and purification of the 20KPEG ShK molecule was asdescribed herein above. The encephalomyelogenic CD4+ rat T cell line,PAS, specific for myelin-basic protein (MBP) originated from Dr. EvelyneBeraud; The maintenance of these cells in vitro and their use in theAT-EAE model has been described earlier [C. Beeton et al. (2001) PNAS98, 13942]. PAS T cells were maintained in vitro by alternating roundsof antigen stimulation or activation with MBP and irradiated thymocytes(2 days), and propagation with T cell growth factors (5 days).Activation of PAS T cells (3×10⁵/ml) involved incubating the cells for 2days with 10 μg/ml MBP and 15×10⁶/ml syngeneic irradiated (3500 rad)thymocytes. On day 2 after in vitro activation, 10-15×10⁶ viable PAS Tcells were injected into 6-12 week old female Lewis rats (Charles RiverLaboratories) by tail IV. Daily subcutaneous injections of vehicle (2%Lewis rat serum in PBS), 20KPEG-ShK or ShK were given from days −1 to 3(FIG. 61), where day −1 represent 1 day prior to injection of PAS Tcells (day 0). In vehicle treated rats, acute EAE developed 4 to 5 daysafter injection of PAS T cells (FIG. 62). Serum was collected byretro-orbital bleeding at day 4 and by cardiac puncture at day 8 (end ofthe study) for analysis of levels of inhibitor. Rats were weighed ondays −1, 4, 6, and 8. Animals were scored blinded once a day from theday of cell transfer (day 0) to day 3, and twice a day from day 4 to day8. Clinical signs were evaluated as the total score of the degree ofparesis of each limb and tail. Clinical scoring: 0=No signs, 0.5=distallimp tail, 1.0=limp tail, 2.0=mild paraparesis, ataxia, 3.0=moderateparaparesis, 3.5=one hind leg paralysis, 4.0=complete hind legparalysis, 5.0=complete hind leg paralysis and incontinence,5.5=tetraplegia, 6.0=moribund state or death. Rats reaching a score of5.5 were euthanized.

Treatment of rats with the Kv1.3 blocker PEG-ShK prior to the onset ofEAE caused a lag in the onset of disease, inhibited the progression ofdisease, and prevented death in a dose-dependent manner (FIG. 62). Onsetof disease in rats that were treated with the vehicle alone, 10 μg/kgShK or 1 μg/kg of PEG-ShK was observed on day 4, compared to day 4.5 inrats treated with 10 μg/kg PEG-ShK or 100 μg/kg PEG-ShK. In addition,rats treated with vehicle alone, 10 μg/kg ShK or 1 μg/kg of PEG-ShK alldeveloped severe disease by the end of the study with an EAE score of5.5 or above. In contrast, rats treated with 10 μg/kg PEG-ShK or 100μg/kg PEG-ShK, reached a peak clinical severity score average of <2, andall but one rat survived to the end of the study. Furthermore, we foundthat rat body weight correlated with disease severity (FIG. 63). Ratstreated with vehicle alone, 10 μg/kg ShK or 1 ug/kg of PEG-ShK all lostan average of 31 g, 30 g, and 30 g, respectively, while rats treatedwith 10 μg/kg PEG-ShK or 100 μg/kg PEG-ShK lost 18 g and 11 g,respectively. Rats in the latter two groups also appeared to be gainingweight by the end of the study, a sign of recovery. It should be notedthat rats treated with 10 μg/kg ShK and 10 μg/kg PEG-ShK received molarequivalents of the ShK peptide. The significantly greater efficacy ofthe PEG-ShK molecule relative to unconjugated ShK, is likely due to thePEG-ShK molecule's greater stability and prolonged half-life in vivo(see, Example 44).

Example 46 Compositions Including Kv1.3 Antagonist Peptides BlockInflammation in Human Whole Blood

Ex vivo assay to examine impact of Kv1.3 inhibitors on secretion of IL-2and IFN-g. Human whole blood was obtained from healthy, non-medicateddonors in a heparin vacutainer. DMEM complete media was Iscoves DMEM(with L-glutamine and 25 mM Hepes buffer) containing 0.1% human albumin(Bayer #68471), 55 μM 2-mercaptoethanol (Gibco), and 1× Pen-Strep-Gln(PSG, Gibco, Cat#10378-016). Thapsigargin was obtained from Alomone Labs(Israel). A 10 mM stock solution of thapsigargin in 100% DMSO wasdiluted with DMEM complete media to a 40 μM, 4× solution to provide the4× thapsigargin stimulus for calcium mobilization. The Kv1.3 inhibitorpeptide ShK (Stichodacytla helianthus toxin, Cat# H2358) and the BKCa1inhibitor peptide IbTx (Iberiotoxin, Cat# H9940) were purchased fromBachem Biosciences, whereas the Kv1.1 inhibitor peptide DTX-k(Dendrotoxin-K) was from Alomone Labs (Israel). The CHO-derivedFc-L10-ShK[2-35] peptibody inhibitor of Kv1.3 was obtained as describedherein at Example 4 and Example 39. The calcineurin inhibitorcyclosporin A was obtained from the Amgen sample bank, but is alsoavailable commercially from a variety of vendors. Ten 3-fold serialdilutions of inhibitors were prepared in DMEM complete media at 4× finalconcentration and 50 μl of each were added to wells of a 96-well Falcon3075 flat-bottom microtiter plate. Whereas columns 1-5 and 7-11 of themicrotiter plate contained inhibitors (each row with a separateinhibitor dilution series), 50 μl of DMEM complete media alone was addedto the 8 wells in column 6 and 100 μl of DMEM complete media alone wasadded to the 8 wells in column 12. To initiate the experiment, 100 μl ofwhole blood was added to each well of the microtiter plate. The platewas then incubated at 37° C., 5% CO₂ for one hour. After one hour, theplate was removed and 50 μl of the 4× thapsigargin stimulus (40 μM) wasadded to all wells of the plate, except the 8 wells in column 12. Theplates were placed back at 37° C., 5% CO₂ for 48 hours. To determine theamount of IL-2 and IFN-g secreted in whole blood, 100 μl of thesupernatant (conditioned media) from each well of the 96-well plate wastransferred to a storage plate. For MSD electrochemilluminesenceanalysis of cytokine production, 20 μl of the supernatants (conditionedmedia) were added to MSD Multi-Spot Custom Coated plates(www.meso-scale.com). The working electrodes on these plates were coatedwith four Capture Antibodies (hIL-5, hIL-2, hIFNg and hIL-4) in advance.After addition of 20 μl of conditioned media to the MSD plate, 150 μl ofa cocktail of Detection Antibodies and P4 Buffer were added to eachwell. The 150 μl cocktail contained 20 ul of four Detection Antibodies(hIL-5, hIL-2, hIFNg and hIL-4) at 1 μg/ml each and 130 ul of 2×P4Buffer. The plates were covered and placed on a shaking platformovernight (in the dark). The next morning the plates were read on theMSD Sector Imager. Since the 8 wells in column 6 of each plate receivedonly the thapsigargin stimulus and no inhibitor, the average MSDresponse here was used to calculate the “High” value for a plate. Thecalculate “Low” value for the plate was derived from the average MSDresponse from the 8 wells in column 12 which contained no thapsigarginstimulus and no inhibitor. Percent of control (POC) is a measure of theresponse relative to the unstimulated versus stimulated controls, where100 POC is equivalent to the average response of thapsigargin stimulusalone or the “High” value. Therefore, 100 POC represents 0% inhibitionof the response. In contrast, 0 POC represents 100% inhibition of theresponse and would be equivalent to the response where no stimulus isgiven or the “Low” value. To calculate percent of control (POC), thefollowing formula is used: [(MSD response ofwell)−(“Low”)]/[(“High”)−(“Low”)]×100. The potency of the molecules inwhole blood was calculated after curve fitting from the inhibition curve(IC) and IC50 was derived using standard curve fitting software.Although we describe here measurement of cytokine production using ahigh throughput MSD electrochemillumenescence assay, one of skill in theart can readily envision lower throughput ELISA assays are equallyapplicable for measuring cytokine production.

Ex vivo assay demonstrating Kv1.3 inhibitors block cell surfaceactivation of CD40L & IL-2R. Human whole blood was obtained fromhealthy, non-medicated donors in a heparin vacutainer. DMEM completemedia was Iscoves DMEM (with L-glutamine and 25 mM Hepes buffer)containing 0.1% human albumin (Bayer #68471), 55 μM 2-mercaptoethanol(Gibco), and 1× Pen-Strep-Gln (PSG, Gibco, Cat#10378-016). Thapsigarginwas obtained from Alomone Labs (Israel). A 10 mM stock solution ofthapsigargin in 100% DMSO was diluted with DMEM complete media to a 40μM, 4× solution to provide the 4× thapsigargin stimulus for calciummobilization. The Kv1.3 inhibitor peptide peptide ShK (Stichodacytlahelianthus toxin, Cat# H2358) and the BKCa1 inhibitor peptide IbTx(iberiotoxin, Cat# H9940) were purchased from Bachem Biosciences,whereas the Kv1.1 inhibitor peptide DTX-k (Dendrotoxin-K) was fromAlomone Labs (Israel). The CHO-derived Fc-L10-ShK[2-35] peptibodyinhibitor of Kv1.3 was obtained as described in Example 4 and Example39. The calcineurin inhibitor cyclosporin A was obtained from the Amgensample bank, but is also available commercially from a variety ofvendors. The ion channel inhibitors ShK, IbTx or DTK-k were diluted intoDMEM complete media to 4× of the final concentration desired (final=50or 100 nM). The calcineurin inhibitor cyclosporin A was also dilutedinto DMEM complete media to 4× final concentration (final=10 μM). Toappropriate wells of a 96-well Falcon 3075 flat-bottom microtiter plate,50 μl of either DMEM complete media or the 4× inhibitor solutions wereadded. Then, 100 μl of human whole blood was added and the plate wasincubated for 1 hour at 37° C., 5% CO₂. After one hour, the plate wasremoved and 50 μl of the 4×thapsigargin stimulus (40 μM) was added toall wells of the plate containing inhibitor. To some wells containing noinhibitor but just DMEM complete media, thapsigargin was also addedwhereas others wells with just DMEM complete media had an additional 50μl of DMEM complete media added. The wells with no inhibitor and nothapsigargin stimulus represented the untreated “Low” control. The wellswith no inhibitor but which received thapsigargin stimulus representedthe control for maximum stimulation or “High” control. Plates wereplaced back at 37° C., 5% CO₂ for 24 hours. After 24 hours, plates wereremoved and wells were process for FACS analysis. Cells were removedfrom the wells and washed in staining buffer (phosphate buffered salinecontaining 2% heat-inactivated fetal calf serum). Red blood cells werelysed using BD FACS Lysing Solution containing 1.5% formaldehyde (BDBiosciences) as directed by the manufacturer. Cells were distributed ata concentration of 1 million cells per 100 microliters of stainingbuffer per tube. Cells were first stained with 1 microliter ofbiotin-labeled anti-human CD4, washed, then stained simultaneously 1microliter each of streptavidin-APC, FITC-labeled anti-human CD45RA, andphycoerythrin (PE)-labeled anti-human CD25 (IL-2Ra) or PE-labeledanti-human CD40L. Cells were washed with staining buffer betweenantibody addition steps. All antibodies were obtained from BDBiosciences (San Diego, Calif.). Twenty to fifty thousand live eventswere collected for each sample on a Becton Dickinson FACSCaliber(Mountain View, Calif.) flow cytometer and analyzed using FlowJosoftware (Tree Star Inc., San Carlos, Calif.). Dead cells, monocytes,and granulocytes were excluded from the analysis on the basis of forwardand side scatter properties.

FIG. 64 and FIG. 67 demonstrate that Kv1.3 inhibitors ShK andFc-L10-ShK[2-35] potently blocked IL-2 secretion in human whole blood,in addition to suppressing activation of the IL-2R on CD4+ T cells. TheKv1.3 inhibitor Fc-L10-ShK[2-35] was more than 200 times more potent inblocking IL-2 production in human whole blood than cyclosporine A (FIG.64) as reflected by the IC50. FIG. 65 shows that Kv1.3 inhibitors alsopotently blocked secretion of IFNg in human whole blood, and FIG. 66demonstrates that upregulation of CD40L on T cells was additionallyblocked. The data in FIGS. 64-67 show that the Fc-L10-ShK[2-35] moleculewas stable in whole blood at 37° C. for up to 48 hours, providing potentblockade of inflammatory responses. Toxin peptide therapeutic agentsthat target Kv1.3 and have prolonged half-life, are sought to providesustained blockade of these responses in vivo over time. In contrast,despite the fact the Kv1.3 inhibitor peptide ShK also showed potentblockade in whole blood, the ShK peptide has a short (˜20 min) half-lifein vivo (C. Beeton et al. (2001) Proc. Natl. Acad. Sci. 98, 13942), andcannot, therefore, provide prolonged blockade. Whole blood represents aphysiologically relevant assay to predict the response in animals. Thewhole blood assays described here can also be used as a pharmacodynamic(PD) assay to measure target coverage and drug exposure following dosingof patients. These human whole blood data support the therapeuticusefulness of the compositions of the present invention for treatment ofa variety immune disorders, such as multiple sclerosis, type 1 diabetes,psoriasis, inflammatory bowel disease, contact-mediated dermatitis,rheumatoid arthritis, psoriatic arthritis, asthma, allergy, restinosis,systemic sclerosis, fibrosis, scleroderma, glomerulonephritis, Sjogrensyndrome, inflammatory bone resorption, transplant rejection,graft-versus-host disease, and lupus.

Example 47 PEGylated Peptibodies

By way of example, PEGylated peptibodies of the present invention weremade by the following method. CHO-expressed FcL10-OsK1 (19.2 mg; MW30,371 Da, 0.63 micromole) in 19.2 ml A5S, 20 mM NaBH₃CN, pH 5, wastreated with 38 mg PEG aldehyde (MW 20 kDa; 3×, Lot 104086). The sealedreaction mixture was stirred in a cold room overnight. The extent of theprotein modification during the course of the reaction was monitored bySEC HPLC using a Superose 6 HR 10/30 column (Amersham Pharmacia Biotech)eluted with a 0.05 M phosphate buffer, 0.5 M NaCl, pH 7.0 at 0.4 ml/min.The reaction mixture was dialyzed with A5S, pH 5 overnight. The dialyzedmaterial was then loaded onto an SP HP FPLC column (16/10) in A5S pH 5and eluted with a 1 M NaCl gradient. The collected fractions wereanalyzed by SEC HPLC, pooled into 3 pools, exchanged into DPBS,concentrated and submitted for functional testing (Table 34).

In another example, FcL10-ShK1 (16.5 mg; MW 30,065 Da, 0.55 micro mole)in 16.5 ml A5S, 20 mM NaBH₃CN, pH 5 was treated with 44 mg PEG aldehyde(MW 20 kDa; 4×, Lot 104086). The sealed reaction mixture was stirred ina cold room overnight. The extent of the protein modification during thecourse of the reaction was monitored by SEC HPLC using a Superose 6 HR10/30 column (Amersham Pharmacia Biotech) eluted with a 0.05 M phosphatebuffer, 0.5 M NaCl, pH 7.0 at 0.4 ml/min. The reaction mixture wasdialyzed with A5S, pH 5 overnight. The dialyzed material was loaded ontoan SP HP FPLC column (16/10) in A5S pH 5 and was eluted with a 1 M NaClgradient. The collected fractions were analyzed by SEC HPLC, pooled into3 pools, exchanged into DPBS, concentrated and submitted for functionaltesting (Table 34).

The data in Table 34 demonstrate potency of the PEGylated peptibodymolecules as Kv1.3 inhibitors.

TABLE 34 shows determinations of IC50 made by whole cell patch clampelectrophysiology with HEK 293 as described in Example 36 herein above.The sustained IC50 was derived from the current 400 msecs after voltageramp from −80 mV to +30 mV. Pool #2 samples comprised di-PEGylatedpeptibodies and Pool #3 samples comprised mono-PEGylated peptibodies.PEGylated Peptibody Pool # IC50 Sustained (nM) PEG-Fc-L10-SHK(2-35) 30.175(n = 4) PEG-Fc-L10-SHK(2-35) 2 0.158(n = 4) PEG-FC-L10-OSK1 30.256(n = 3) PEG-FC-L10-OSK1 2 0.332(n = 3)

Example 48 PEGylated Toxin Peptides

Shk and Osk-1 PEGylation, purification and analysis. Synthetic Shk orOSK1-1 toxin peptides were selectively PEGylated by reductive alkylationat their N-termini. Conjugation was achieved, with either Shk or OSK-1toxin peptides, at 2 mg/ml in 50 mM NaH₂PO₄, pH 4.5 reaction buffercontaining 20 mM sodium cyanoborohydride and a 2 molar excess of 20 kDamonomethoxy-PEG-aldehyde (Nektar Therapeutics, Huntsville, Ala.).Conjugation reactions were stirred overnight at room temperature, andtheir progress was monitored by RP-HPLC. Completed reactions werequenched by 4-fold dilution with 20 mM NaOAc, pH 4, adjusted to pH 3.5and chilled to 4° C. The PEG-peptides were then purifiedchromatographically at 4° C.; using SP Sepharose HP columns (GEHealthcare, Piscataway, N.J.) eluted with linear 0-1M NaCl gradients in20 mM NaOAc, pH 4.0. (FIG. 68A and FIG. 68B) Eluted peak fractions wereanalyzed by SDS-PAGE and RP-HPLC and pooling determined by purity >97%.Principle contaminants observed were di-PEGylated toxin peptide andunmodified toxin peptide. Selected pools were concentrated to 2-5 mg/mlby centrifugal filtration against 3 kDa MWCO membranes and dialyzed into10 mM NaOAc, pH 4 with 5% sorbitol. Dialyzed pools were then sterilefiltered through 0.2 micron filters and purity determined to be >97% bySDS-PAGE and RP-HPLC (FIG. 69A and FIG. 69B). Reverse-phase HPLC wasperformed on an Agilent 1100 model HPLC running a Zorbax 5 μm 300SB-C84.6×50 mm column (Phenomenex) in 0.1% TFA/H₂O at 1 ml/min and columntemperature maintained at 40° C. Samples of PEG-peptide (20 μg) wereinjected and eluted in a linear 6-60% gradient while monitoringwavelengths 215 nm and 280 nm.

Electrophysiology performed by patch clamp on whole cells (see, Example36) yielded a peak IC50 of 1.285 nM for PEG-OSK1 and 0.169 nM forPEG-ShK[1-35] (FIG. 74), in a concentration dependent block of theoutward potassium current recorded from HEK293 cells stably expressinghuman Kv1.3 channel. The purified PEG-ShK[1-35] molecule, also referredto as “20K PEG-ShK[1-35]” and “PEG-ShK”, had a much longer half-life invivo than the small ShK peptide (FIG. 59 and FIG. 60). PEG-ShK[1-35]suppressed severe autoimmune encephalomyelitis in rats (Example 45,FIGS. 61-63) and showed greater efficacy than the small native ShKpeptide.

Example 49 Fc Loop Insertions of ShK and OSK1 Toxin Peptides

As exemplified in FIG. 70, FIG. 71, FIG. 72, and FIG. 73,disulphide-constrained toxin peptides were inserted into the human IgG1Fc-loop domain, defined as the sequence D₁₃₇E₁₃₈L₁₃₉T₁₄₀K₁₄₁, accordingto the method published in Example 1 in Gegg et al., Modified Fcmolecules, WO 2006/036834 A2 [PCT/US2005/034273]). ExemplaryFcLoop-L2-OsK1-L2, FcLoop-L2-ShK-L2, FcLoop-L2-ShK-L4, andFcLoop-L4-OsK1-L2 were made having three linked domains. These werecollected, purified and submitted for functional testing.

The peptide insertion for these examples was between Fc residues Leu₁₃₉and Thr₁₄₀ and included 2-4 Gly residues as linkers flanking either sideof the inserted peptide. However, alternate insertion sites for thehuman IgG1 Fc sequence, or different linkers, are also useful in thepractice of the present invention, as is known in the art, e.g., asdescribed in Example 13 of Gegg et al., Modified Fc molecules, WO2006/036834 A2 [PCT/US2005/034273]).

Example 50 Purification of ShK(2-35)-L-Fc from E. coli

Frozen, E. coli paste (117 g), obtained as described in Example 16herein above, was combined with 1200 ml of room temperature 50 mM trisHCl, 5 mM EDTA, pH 7.5 and was brought to about 0.1 mg/ml hen egg whitelysozyme. The suspended paste was passed through a chilledmicrofluidizer twice at 12,000 PSI. The cell lysate was then centrifugedat 17,700 g for 30 min at 4° C. The pellet was then resuspended in 1200ml 1% deoxycholic acid using a tissue grinder and then centrifuged at17,700 g for 30 min at 4° C. The pellet was then resuspended in 1200 mlwater using a tissue grinder and then centrifuged at 17,700 g for 30 minat 4° C. 6.4 g of the pellet (total 14.2g) was then dissolved in 128 ml8 M guanidine HCl, 50 mM tris HCl, pH 8.0. 120 ml of the pellet solutionwas then incubated with 0.67 ml of 1 M DTT for 60 min at 37° C. Thereduced material was transferred to 5500 ml of the refolding buffer (3 Murea, 50 mM tris, 160 mM arginine HCl, 2.5 mM EDTA, 2.5 mM cystamineHCl, 4 mM cysteine, pH 9.5) at 2 ml/min, 4° C. with vigorous stirring.The stirring rate was then slowed and the incubation was continued for 3days at 4° C.

The refold was diluted with 5.5 L of water, and the pH was adjusted to8.0 using acetic acid, then the solution was filtered through a 0.22 μmcellulose acetate filter and loaded on to a 35 ml Amersham QSepharose-FF (2.6 cm I.D.) column at 10 ml/min in Q-Buffer A (20 mMTris, pH 8.5) at 8° C. with an inline 35 ml Amersham Mab Select column(2.6 cm I.D.). After loading, the Q Sepharose column was removed fromthe circuit, and the remaining chromatography was carried out on the MabSelect column. The column was washed with several column volumes ofQ-Buffer A, followed by elution using a step to 100 mM glycine pH 3.0.The fractions containing the desired product immediately loaded on to a5.0 ml Amersham SP-Sepharose HP column at 5.0 ml/min in S-Buffer A (10mM NaH₂PO₄, pH 7.0) at 8° C. The column was then washed with severalcolumn volumes of S-Buffer A followed by a linear gradient from 5% to60% S-Buffer B (10 mM NaH₂PO₄, 1 M NaCl, pH 7.0) followed by a step to100% S-Buffer B. Fractions were then analyzed using a Coomassiebrilliant blue stained tris-glycine 4-20% SDS-PAGE. The fractionscontaining the bulk of the desired product were pooled and then appliedto a 50 ml MEP Hypercel column (2.6 cm I.D.) at 10 ml/min in MEP BufferA (20 mM tris, 200 mM NaCl, pH 8.0) at 8° C. Column was eluted with alinear gradient from 5% to 50% MEP Buffer B (50 mM sodium citrate pH4.0) followed by a step to 100% MEP Buffer B. Fractions were thenanalyzed using a Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE, and the fractions containing the bulk of the desired productwere pooled.

The MEP-pool was then concentrated to about 10 ml using a Pall Jumbo-Sepwith a 10 kDa membrane. A spectral scan was then conducted on 50 μl ofthe combined pool diluted in 700 μl PBS using a Hewlett Packard 8453spectrophotometer (FIG. 76A). Then concentration of the material wasdetermined to be 3.7 mg/ml using a calculated molecular mass of 30,253and extinction coefficient of 36,900 M⁻¹ cm⁻¹. The purity of thematerial was then assessed using a Coomassie brilliant blue stainedtris-glycine 4-20% SDS-PAGE (FIG. 76B). The macromolecular state of theproduct was then determined using size exclusion chromatography on 70 μgof the product injected on to a Phenomenex BioSep SEC 3000 column(7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observingthe absorbance at 280 nm (FIG. 76C). The product was then subject tomass spectral analysis by chromatographing approximately 4 μg of thesample through a RP-HPLC column (Vydac C₄, 1×150 mm). Solvent A was 0.1%trifluoroacetic acid in water and solvent B was 0.1% trifluoroaceticacid in 90% acetonitrile, 10% water. The column was pre-equilibrated in10% solvent B at a flow rate of 80 μl per min. The protein was elutedusing a linear gradient of 10% to 90% solvent B over 30 min. Part of theeffluent was directed into a LCQ ion trap mass spectrometer. The massspectrum was deconvoluted using the Bioworks software provided by themass spectrometer manufacturer. (FIG. 76D). The product was filteredthrough a 0.22 μm cellulose acetate filter and then stored at −80° C.

In Table 35, IC50 data for the purified E. coli-derived ShK[2-35]-L-Fcare compared to some other embodiments of the inventive composition ofmatter.

TABLE 35 E. coli-derived recombinant Fc-L-ShK[1-35], Fc-L-ShK[2-35],Fc-L-OSK1, Shk[1-35]-L-Fc and ShK[2-35]-L-Fc peptibodies containing Fcat either the N-terminus or C-terminus show potent blockade of humanKv1.3. The activity of the CHO-derived Fc-L10- ShK[1-35] R1Q mutant isalso shown. Whole cell patch clamp electrophysiology (WCVC), by methodsdescribed in Example 36, was performed using HEK293/Kv1.3 cells and theIC50 shown is the average from dose-response curves from 3 or morecells. IonWorks ™ (IWQ) planar patch clamp electrophysiology by methodsdescribed in Example 44 was on CHO/Kv1.3 cells and the average IC50 isshown. The inventive molecules were obtained by methods as described inthe indicated Example: E. coli-derived Fc-L-ShK[1-35] (Example 3 andExample 38), E. coli-derived Fc-L-ShK[2-35] (Example 4 and Example 39),E. coli Fc-L-OSK1 (Example 10 and Example 40), ShK[1-35]-L-Fc (Example15 and Example 51), and ShK[2-35]-L-Fc (Example 16 and this Example 50).CHO-derived Fc-L10-ShK[1-35] R1Q molecule was generated using methodssimilar to those described for CHO-derived Fc-L10-ShK[1-35], Kv1.3 IC₅₀Kv1.3 IC₅₀ Molecule by WCVC (nM) by IWQ (nM) E. coli-derivedFc-L-ShK[1-35] 1.4 E. coli-derived Fc-L-ShK[2-35] 1.3 2.8 E.coli-derived Fc-L-OSK1 3.2 E. coli-derived Shk[1-35]-L-Fc 2.4 E.coli-derived ShK[2-35]-L-Fc 4.9 CHO-derived Fc-L10-ShK[1-35] 2.2 R1Q

Example 51 Purification of Met-ShK(1-35)-Fc from E. coli

Frozen, E. coli paste (65 g), obtained as described in Example 15 hereinabove was combined with 660 ml of room temperature 50 mM tris HCl, 5 mMEDTA, pH 7.5 and was brought to about 0.1 mg/ml hen egg white lysozyme.The suspended paste was passed through a chilled microfluidizer twice at12,000 PSI. The cell lysate was then centrifuged at 17,700 g for 30 minat 4° C. The pellet was then resuspended in 660 ml 1% deoxycholic acidusing a tissue grinder and then centrifuged at 17,700 g for 30 min at 4°C. The pellet was then resuspended in 660 ml water using a tissuegrinder and then centrifuged at 17,700 g for 30 min at 4° C. 13 g of thepellet was then dissolved in 130 ml 8 M guanidine HCl, 50 mM tris HCl,pH 8.0. 10 ml of the pellet solution was then incubated with 0.1 ml of 1M DTT for 60 min at 37° C. The reduced material was transferred to 1000ml of the refolding buffer (2 M urea, 50 mM tris, 160 mM arginine HCl,2.5 mM EDTA, 1.2 mM cystamine HCl, 4 mM cysteine, pH 8.5) at 2 ml/min,4° C. with vigorous stirring. The stirring rate was then slowed and theincubation was continued for 3 days at 4° C.

The refold was diluted with 1 L of water, and filtered through a 0.22 μmcellulose acetate filter then loaded on to a 35 ml Amersham QSepharose-FF (2.6 cm I.D.) column at 10 ml/min in Q-Buffer A (20 mMTris, pH 8.5) at 8° C. with an inline 35 ml Amersham Mab Select column(2.6 cm I.D.). After loading, the Q Sepharose column was removed fromthe circuit, and the remaining chromatography was carried out on the MabSelect column. The column was washed with several column volumes ofQ-Buffer A, followed by elution using a step to 100 mM glycine pH 3.0.The fractions containing the desired product immediately loaded on to a5.0 ml Amersham SP-Sepharose HP column at 5.0 ml/min in S-Buffer A (20mM NaH₂PO₄, pH 7.0) at 8° C. The column was then washed with severalcolumn volumes of S-Buffer A followed by a linear gradient from 5% to60% S-Buffer B (20 mM NaH₂PO₄, 1 M NaCl, pH 7.0) followed by a step to100% S-Buffer B. Fractions were then analyzed using a Coomassiebrilliant blue stained tris-glycine 4-20% SDS-PAGE. The fractionscontaining the bulk of the desired product were pooled.

The S-pool was then concentrated to about 10 ml using a Pall Jumbo-Sepwith a 10 kDa membrane. A spectral scan was then conducted on 20 μl ofthe combined pool diluted in 700 μl PBS using a Hewlett Packard 8453spectrophotometer (FIG. 77A). Then concentration of the material wasdetermined to be 3.1 mg/ml using a calculated molecular mass of 30,409and extinction coefficient of 36,900 M⁻¹ cm⁻¹. The purity of thematerial was then assessed using a Coomassie brilliant blue stainedtris-glycine 4-20% SDS-PAGE (FIG. 77B). The macromolecular state of theproduct was then determined using size exclusion chromatography on 93 μgof the product injected on to a Phenomenex BioSep SEC 3000 column(7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observingthe absorbance at 280 nm (FIG. 77C). The product was then subject tomass spectral analysis by MALDI mass spectrometry. An aliquot of thesample was spotted with the MALDI matrix sinapinic acid on sample plate.A Voyager DE-RP time-of-flight mass spectrometer equipped with anitrogen laser (337 nm, 3 ns pulse) was used to collect spectra. Thepositive ion/linear mode was used, with an accelerating voltage of 25kV. Each spectrum was produced by accumulating data from ˜200 lasershots (FIG. 77D). External mass calibration was accomplished usingpurified proteins of known molecular masses.

The IC₅₀ for blockade of human Kv1.3 by purified E. coli-derivedMet-ShK(1-35)-Fc, also referred to as “ShK[1-35]-L-Fc”, is shown inTable 35 herein above.

Example 52 Bacterial Expression of OsK1-L-Fc Inhibitor of Kv1.3

The methods to clone and express the peptibody in bacteria were asdescribed in Example 3. The vector used was pAMG21amgR-pep-Fc and theoligos listed below were used to generate a duplex (see below) forcloning and expression in bacteria of OsK1-L-Fc.

Oligos used to form duplex are shown below:

GGGTGTTATCATCAACGTTAAATGCAAAATCTCCCGTCAGTGCCTGGAACCGTGCAAAAAAGCTGGTATGCGT;//SEQ ID NO: 1347TTCGGTAAATCCATGAACGGTAAATGCCACTGCACCCCGAAATCTGGTGGTGGTGGTTCT;//SEQ ID NO: 1348CACCAGAACCACCACCACCACCAGATTTCGGGGTGCAGTGGCATTTACCGTTCATGCATTTACCGAAACGCAT;//SEQ ID NO: 1349ACCAGCTTTTTTGCACGGTTCCAGGCACTGACGGGAGATTTTGCATTTAACGTTGATGATAAC;//SEQ ID NO: 1310The oligos shown above were used to form the duplex shown below:

GGGTGTTATCATCAACGTTAAATGCAAAATCTCCCGTCAGTGCCTGGAACCGTGCAAAAA//SEQ ID NO: 1350 1---------+---------+---------+---------+---------+---------+  60    CAATAGTAGTTGCAATTTACGTTTTAGAGGGCAGTCACGGACCTTGGCACCTTTTT//SEQ ID NO: 1352 G  V  I  I  N  V  K  C  K  I  S  R  Q  C  L  E  P  C  K  K -//SEQ ID NO: 1351AGCTGGTATGCGTTTCGGTAAATGCATGAACGGTAAATGCCACTGCACCCCGAAATCTGG 61---------+---------+---------+---------+---------+---------+ 120TCGACCATACGCAAAGCCATTTACGTACTTGCCATTTACGGTGACGTGGGGCTTTAGACC A  G  M  R  F  G  K  C  M  N  G  K  C  H  C  T  P  K  S  G -TGGTGGTGGTTCT 121 ---------+------- 137 ACCACCACCAAGACCAC  G  G  G  S  G-

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 53 Bacterial Expression of Gly-ShK(1-35)-L-Fc Inhibitor of Kv1.3

The methods to clone and express the peptibody in bacteria were asdescribed in Example 3. The vector used was pAMG21amgR-pep-Fc and theoligos listed below were used to generate a duplex (see below) forcloning and expression in bacteria of Gly-ShK(1-35)-L-Fc. Oligos used toform duplex are shown below:

GGGTCGTTCTTGTATTGATACTATTCCAAAATCTCGTTGTACTGCTTTTCAATGTAAACATTCTA//SEQ ID NO: 1313 TGAAATATCGTCTTTCTT;TTTGTCGTAAAACTTGTGGTACTTGTTCTGGTGGTGGTGGTTCT; //SEQ ID NO: 1314CACCAGAACCACCACCACCAGAACAAGTACCACAAGTTTTACGACAAAAAGAAAGACGATATTT//SEQ ID NO: 1353 CATAGAATGTTTACATTGA;AAAGCAGTACAACGAGATTTTGGAATAGTATCAATACAAGAACG //SEQ ID NO: 1354

The oligos shown above were used to form the duplex shown below:

GGGTCGTTCTTGTATTCATACTATTCCAAAATCTCGTTGTACTGCTTTTCAATGTAAACA//SEQ ID NO: 1355 1---------+---------+---------+---------+---------+---------+  60    GCAAGAACATAACTATGATAAGGTTTTAGAGCAACATGACGAAAAGTTACATTTGT//SEQ ID NO: 1357 G  R  S  C  I  D  T  I  P  K    S  R  C  T  A  F  Q  C  K  H   -//SEQ ID NO: 1356TTCTATGAAATATCGTCTTTCTTTTTGTCGTAAAACTTGTGGTACTTGTTCTGGTGGTGG 61---------+---------+---------+---------+---------+---------+ 120AAGATACTTTATAGCAGAAAGAAAAACAGCATTTTGAACACCATGAACAAGACCACCACC S  M  K  Y  R  L  S  F  C  R  K  T  C  G  T  C  S  G  G  G   - TGGTTCT121 ---------+- 131 ACCAAGACCAC  G  S  G -

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Abbreviations

Abbreviations used throughout this specification are as defined below,unless otherwise defined in specific circumstances.

Ac acetyl (used to refer to acetylated residues)

AcBpa acetylated p-benzoyl-L-phenylalanine

ADCC antibody-dependent cellular cytotoxicity

Aib aminoisobutyric acid

bA beta-alanine

Bpa p-benzoyl-L-phenylalanine

BrAc bromoacetyl (BrCH₂C(O)

BSA Bovine serum albumin

Bzl Benzyl

Cap Caproic acid

COPD Chronic obstructive pulmonary disease

CTL Cytotoxic T lymphocytes

DCC Dicylcohexylcarbodiimide

Dde 1-(4,4-dimethyl-2,6-dioxo-cyclohexylidene)ethyl

ESI-MS Electron spray ionization mass spectrometry

Fmoc fluorenylmethoxycarbonyl

HOBt 1-Hydroxybenzotriazole

HPLC high performance liquid chromatography

HSL homoserine lactone

IB inclusion bodies

KCa calcium-activated potassium channel (including IKCa, BKCa, SKCa)

Kv voltage-gated potassium channel

Lau Lauric acid

LPS lipopolysaccharide

LYMPH lymphocytes

MALDI-MS Matrix-assisted laser desorption ionization mass spectrometry

Me methyl

MeO methoxy

MHC major histocompatibility complex

MMP matrix metalloproteinase

1-Nap 1-napthylalanine

NEUT neutrophils

Nle norleucine

NMP N-methyl-2-pyrrolidinone

PAGE polyacrylamide gel electrophoresis

PBMC peripheral blood mononuclear cell

PBS Phosphate-buffered saline

Pbf 2,2,4,6,7-pendamethyldihydrobenzofuran-5-sulfonyl

PCR polymerase chain reaction

Pec pipecolic acid

PEG Poly(ethylene glycol)

pGlu pyroglutamic acid

Pic picolinic acid

pY phosphotyrosine

RBS ribosome binding site

RT room temperature (25° C.)

Sar sarcosine

SDS sodium dodecyl sulfate

STK serine-threonine kinases

t-Boc tert-Butoxycarbonyl

tBu tert-Butyl

THF thymic humoral factor

Trt trityl

1. A composition of matter of the formula(X¹)a—(F¹)d—(X²)b—(F²)e—(X³)c or multimers thereof, wherein: F¹ and F²are half-life extending moieties, and d and e are each independently 0or 1, provided that at least one of d and e is 1; X¹, X², and X³ areeach independently -(L)_(f)-P-(L)₉-, and f and g are each independently0 or 1; P is a ShK peptide analog of no more than about 80 amino acidresidues in length, comprising the amino acid sequence of SEQ ID NO:914, SEQ ID NO: 915, SEQ ID NO: 916, or SEQ ID NO: 917, and comprisingat least two intrapeptide disulfide bonds; L is a linker; and a, b, andc are each independently 0 or 1, provided that at least one of a, b andc is 1; or a physiologically acceptable salt thereof.
 2. The compositionof matter of claim 1 of the formula P-(L)_(g)-F¹.
 3. The composition ofmatter of claim 1 of the formula F¹-(L)_(f)-P.
 4. The composition ofmatter of claim 1 of the formula P-(L)_(g)-F¹-(L)_(f)-P.
 5. Thecomposition of matter of claim 1 of the formula F¹-(L)_(f)-P-(L)_(g)-F².6. The composition of matter of claim 1 of the formulaF¹-(L)_(f)-P-(L)_(g)-F²-(L)_(f)-P.
 7. The composition of matter of claim1 of the formula F¹-F²-(L)_(f)-P
 8. The composition of matter of claim 1of the formula P-(L)_(g)-F¹-F².
 9. The composition of matter of claim 1of the formula P-(L)_(g)-F¹-F²-(L)_(f)-P.
 10. The composition of matterof claim 1, wherein F¹ or F², or both is a copolymer of ethylene glycol,a polypropylene glycol, a copolymer of propylene glycol, acarboxymethylcellulose, a polyvinyl pyrrolidone, a poly-1,3-dioxolane, apoly-1,3,6-trioxane, an ethylene/maleic anhydride copolymer, apolyaminoacid, a dextran n-vinyl pyrrolidone, a poly n-vinylpyrrolidone, a propylene glycol homopolymer, a propylene oxide polymer,an ethylene oxide polymer, a polyoxyethylated polyol, a polyvinylalcohol, a linear or branched glycosylated chain, a polyacetal, a longchain fatty acid, a long chain hydrophobic aliphatic group, animmunoglobulin F_(c) domain or portion thereof, a CH2 domain of Fc, anFc domain loop, an albumin, an albumin-binding protein, a transthyretin,a thyroxine-binding globulin, or a ligand that has an affinity for along half-life serum protein, said ligand being selected from the groupconsisting of peptide ligands and small molecule ligands; or acombination of any of these members.
 11. The composition of matter ofclaim 1 wherein F¹ or F², or both, comprises a human IgG Fc domain or aportion thereof.
 12. The composition of matter of claim 11, wherein thehuman IgG. Fc domain comprises a human IgG1 Fc domain.
 13. Thecomposition of matter of claim 11, wherein the human IgG Fc domaincomprises a human IgG2 Fc domain.
 14. The composition of matter of claim1, wherein F¹ and F² are different half-life extending moieties.
 15. Thecomposition of matter of claim 1, wherein F¹ or F², or both, comprises asequence selected from the group consisting of SEQ ID NOS: 2, 4, 70, 71,72, 74, 75, 76, 1340 through 1342, and 1359 through 1363 as set forth in(FIGS. 3, 4, 11A-C, 12A-C, and 12E-F).
 16. The composition of matter ofclaim 1, wherein F¹ or F², or both, comprises a human serum albuminprotein domain.
 17. The composition of matter of claim 1, wherein F orF², or both, comprises a transthyretin protein domain.
 18. Thecomposition of matter of claim 1, wherein F¹ or F², or both, comprises abiologically suitable polymer or copolymer.
 19. The composition ofmatter of claim 18, wherein the biologically suitable polymer ispolyethylene glycol (PEG) of molecular mass about 1,000 Da to about100,000 Da.
 20. The composition of matter of claim 19, wherein the PEGis selected from the group consisting of 5 kD and 20 kD PEG.
 21. Thecomposition of matter of any of claim 5, 6, 8, or 9, wherein F¹ is anhuman IgG Fc domain or HSA, and F² is PEG.
 22. The composition of matterof any of claim 5, 6, 7 or 9, wherein F² is an human IgG Fc domain orHSA and F¹ is PEG.
 23. The composition of matter of claim 1, furthercomprising one or more PEG moieties conjugated to a non-PEG F¹ ornon-PEG F², or to P, or to any combination of any of these.
 24. Thecomposition of matter of claim 1, in which the toxin peptide is insertedinto a human IgG1 Fc domain loop.
 25. A composition of matter,comprising a ShK peptide analog that comprises an amino acid sequenceselected from the group consisting of SEQ ID NOS: 914 through 917 as setforth in Table 2, or a physiologically acceptable salt thereof.
 26. Apharmaceutical composition, comprising the composition of claim 1 orclaim 25; and a pharmaceutically acceptable carrier.
 27. The compositionof matter of claim 1, wherein any f or any g is 1, and L is a peptidelinker comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 79, 84, and 637 through
 656. 28. Thecomposition of matter of claim 1 or claim 25, comprising a ShK peptideanalog that comprises the amino acid sequence of SEQ ID NO:
 914. 29. Thecomposition of matter of claim 1 or claim 25, comprising a ShK peptideanalog that comprises the amino acid sequence of SEQ ID NO:
 915. 30. Thecomposition of matter of claim 1 or claim 25, comprising a ShK peptideanalog that comprises the amino acid sequence of SEQ ID NO:
 916. 31. Thecomposition of matter of claim 1 or claim 25, comprising a ShK peptideanalog that comprises the amino acid sequence of SEQ ID NO: 917.